Devices and methods for determining the amount of energy absorbed during irradiation

ABSTRACT

Devices and methods are disclosed for determining the amount of energy absorbed during irradiation. Such devices comprise a material that absorbs radiation in a quantifiable manner and a cooling agent to maintain the temperature of that material within a predetermined range, and may be used to determine the amount of energy absorbed during irradiation, for example during sterilization of a biological material.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to devices and methods fordetermining the amount of energy absorbed during irradiation. Thepresent invention particularly relates to devices comprising a materialthat absorbs radiation in a quantifiable manner and a cooling agent tomaintain the temperature of that material within a predetermined range,and the use of these devices for determining the amount of energyabsorbed during irradiation, for example during sterilization of abiological material.

[0003] 2. Background of the Related Art

[0004] Many biological materials that are prepared for human,veterinary, diagnostic and/or experimental use may contain unwanted andpotentially dangerous biological contaminants or pathogens, such asviruses, bacteria, in both vegetative and spore states, yeasts, molds,fungi, prions or similar agents responsible, alone or in combination,for TSEs and/or single-cell or multicellular parasites. Consequently, itis of utmost importance that any biological contaminant or pathogen inthe biological material be inactivated before the product is used. Thisis especially critical when the material is to be administered directlyto a patient, for example in blood transfusions, blood factorreplacement therapy, tissue implants, including organ transplants, andother forms of human and/or other animal therapy corrected or treated bysurgical implantation, intravenous, intramuscular or other forms ofinjection or introduction. This is also critical for the variousbiological materials that are prepared in media or via the culture ofcells, or recombinant cells which contain various types of plasma and/orplasma derivatives or other biologic materials and which may be subjectto mycoplasmal, prion, ureaplasmal, bacterial, viral and/or otherbiological contaminants or pathogens.

[0005] In order to inactivate such biological contaminants or pathogens,it is often desirable to expose the biological material to radiation.For instance, viruses and bacteria are readily inactivated by gammaradiation at high total doses.

[0006] In order to determine the amount of radiation received by abiological material during treatment with radiation, dosimetry isemployed. Dosimetry is the part of a radiation process where the amountof energy absorbed during irradiation is quantified. Dosimetry isemployed, for instance, to correctly monitor radiation processes duringthe development, validation and routine process control stages.

[0007] For instance, when irradiating biological materials to inactivatebiological contaminants and pathogens, dosimetry may be used todetermine whether the amount of radiation received by the biologicalproduct during irradiation is within a predetermined range. If theamount of radiation received is below a predetermined amount, thecontaminants or pathogens in the biological material may not beinactivated. Conversely, if the amount of radiation received is above apredetermined amount, the biological material may lose biologicalactivity. In either case, the biological material would be unsuitablefor use.

[0008] Dosimeters are devices that, when irradiated, exhibit aquantifiable and reproducible change in some property of the device thatmay be related to absorbed dose in a given material. Physical and/orchemical changes take place in the device than can be measured usingappropriate analytical instrumentation and techniques.

[0009] Examples of solid state dosimeters include thermoluminescentdosimeters, lyoluminescent dosimeters, polymethyl methacrylatedosimeters, radiochromicatic film dosimeters, cobalt glass dosimetersand alanine dosimeters. Dosimeters may have various geometries, such aspellets, films and cylinders.

[0010] In practice, the dosimeter is irradiated and the amount ofradiation received is determined by measuring a characteristic of thedosimeter sensitive to the radiation. For instance, depending on thetype of dosimeter employed, luminescence, absorption, or free radicalgeneration may be measured.

[0011] When the crystalline form of alanine is irradiated, stable,characteristic free radicals are produced. The number of free radicalsis proportional to the radiation dose absorbed by the crystal. Bydetermining the amount of free radicals produced during irradiation, thedose of radiation received may be determined. For alanine dosimeters,the amount of free radicals produced is typically determined by electronspin resonance spectroscopy (ESR). The free radicals generated duringirradiation of alanine remain stable; their concentration is subject toonly a minor amount of time-dependent change. Additionally, the freeradicals generated in crystalline alanine are relatively stable withrespect to heat. Examples of alanine dosimeters are disclosed in U.S.Pat. Nos. 4,668,714 and 5,066,863.

[0012] Dosimeters may also be used for dose mapping. The process of dosemapping typically includes simulating radiation conditions to beemployed for a sample of interest. A dosimeter is fixed in, on or near asimulated sample, the simulated sample is irradiated and the amount ofradiation received by the dosimeter during irradiation is determined.The amount of radiation received by the dosimeter corresponds to theamount received by the simulated sample.

[0013] U.S. Pat. No. 6,157,028 to Purtle discloses a method of dosemapping. According to Purtle, a dosimeter is packed inside a containerunder conditions that simulate the conditions under which a material ofinterest is to be irradiated, e.g., the density of the material withinthe container approximates the density of the material to be irradiated,the relationship between the container and the radiation sourceapproximates that to be used during irradiation of the material ofinterest, etc. A mixture of dry animal food and salt pelletsapproximating the density of dry ice is packed around the container andthe container is irradiated. Following irradiation, the dosimeter isanalyzed.

[0014] Purtle teaches that biological materials are typically irradiatedat temperatures below ambient, but states that a dry ice substitute isemployed during dose mapping because “[d]osimeters . . . do not giveaccurate data in cold conditions . . . ” In addition to the dry icesubstitute, a material other than the material of interest is employed,such as agar.

[0015] The above references are incorporated by reference herein whereappropriate for teachings of additional or alternative details, featuresand/or technical background.

SUMMARY OF THE INVENTION

[0016] An object of the invention is to solve at least the related artproblems and disadvantages, and to provide at least the advantagesdescribed hereinafter.

[0017] Accordingly, it is an object of the present invention to providemethods for determining the amount of energy absorbed by a productundergoing sterilization with radiation.

[0018] Accordingly, it is another object of the present invention toprovide devices for measuring the amount of energy absorbed by a productundergoing irradiation.

[0019] Another object of the present invention is to provide a methodfor maintaining the temperature of a product undergoing irradiationwithin a predetermined temperature range.

[0020] In accordance with these and other objectives, a first embodimentof the present invention is directed to a device for measuring theamount of energy absorbed by a product undergoing irradiation,comprising: (i) an effective amount of at least one material thatabsorbs radiation in a quantifiable manner; and (ii) an effective amountof at least one cooling agent for maintaining the material within apredetermined temperature range between −120° C. and ambient temperatureduring irradiation.

[0021] A second embodiment of the present invention is directed to amethod for determining the amount of energy absorbed by a productundergoing irradiation, comprising: (a) placing within a suitablecontainer at least one product to be irradiated and at least one devicecomprising: (i) at least one material that absorbs radiation in aquantifiable manner; and (ii) an effective amount of at least onecooling agent for maintaining the material within a predeterminedtemperature range between −120° C. and ambient temperature duringirradiation; (b) irradiating the container containing the product andthe device; and (c) analyzing the material to determine the amount ofenergy absorbed during irradiation.

[0022] A third embodiment of the present invention is directed to amethod for maintaining the temperature of a product undergoingirradiation within a predetermined temperature range between −120° C.and ambient temperature, comprising: (a) placing at least one product tobe irradiated in a suitable container having at least one side and abottom, wherein the volume defined by said container is greater than thevolume of the product; (b) placing an effective amount of at least onecooling agent in the container between the product and the at least oneside; and (c) irradiating the container containing the product and thecooling agent with ionizing radiation.

[0023] Other objects, features and advantages of the present inventionwill be set forth in the detailed description of preferred embodimentsthat follows, and in part will be apparent from the description or maybe learned by practice of the invention. These objects and advantages ofthe invention will be realized and attained by the compositions andmethods particularly pointed out in the written description and claimshereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024]FIG. 1 shows a device according to a preferred embodiment of thepresent invention.

[0025]FIG. 2 shows the placement of alanine dosimeters in a deviceaccording to a preferred embodiment of the present invention.

[0026]FIG. 3 shows the placement of alanine dosimeters and thermal tapeon a device according to a preferred embodiment of the presentinvention.

[0027]FIG. 4 shows the comparability of average measurements for alaninedosimeters irradiated with gamma irradiation taken over time.

[0028]FIG. 5 shows a plot of mass-corrected dosimeter response versusabsorbed dose over a calibration range for alanine dosimeters irradiatedwith gamma irradiation to various total doses.

[0029]FIG. 6 shows a calibration curve (T=25° C.) for an alaninedosimeter irradiated with gamma irradiation to total doses of 25 to 115kGy.

[0030]FIG. 7 shows a plot of relative response versus temperature foralanine dosimeters irradiated with gamma radiation between −77 and 50°C. to various total doses.

[0031]FIG. 8 shows a plot or relative response versus temperature foralanine dosimeters irradiated with gamma irradiation between −77 and 50°C. to various total doses.

[0032]FIG. 9 shows the comparison of computed dose to reported dose foralanine dosimeters irradiated with gamma irradiation between −77 and 50°C. to various total doses.

[0033]FIG. 10 shows response as a function of temperature for alaninedosimeters irradiated with gamma irradiation between −10 and 50° C. tovarious total doses.

[0034]FIG. 11 shows a scatter plot (response versus dose) at dry icetermperature over the dose range of 25 to 100 kGy.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS A. Definitions

[0035] Unless defined otherwise, all technical and scientific terms usedherein are intended to have the same meaning as is commonly understoodby one of ordinary skill in the relevant art.

[0036] As used herein, the singular forms “a,” “an,” and “the” includethe plural reference unless the context clearly dictates otherwise.

[0037] As used herein, the term “biological material” is intended tomean any substance derived or obtained from a living organism.Illustrative examples of biological materials include, but are notlimited to, the following: cells; tissues; blood or blood components;proteins, including recombinant and transgenic proteins, andproteinaceous materials; enzymes, including digestive enzymes, such astrypsin, chymotrypsin, alpha-glucosidase and iduronodate-2-sulfatase;immunoglobulins, including mono and polyimmunoglobulins; botanicals;food; and the like. Preferred examples of biological materials include,but are not limited to, the following: ligaments; tendons; nerves; bone,including demineralized bone matrix, grafts, joints, femurs, femoralheads, etc.; teeth; skin grafts; bone marrow, including bone marrow cellsuspensions, whole or processed; heart valves; cartilage; corneas;arteries and veins; organs, including organs for transplantation, suchas hearts, livers, lungs, kidneys, intestines, pancreas, limbs anddigits; lipids; carbohydrates; collagen, including native, afibrillar,atelomeric, soluble and insoluble, recombinant and transgenic, bothnative sequence and modified; enzymes; chitin and its derivatives,including NO-carboxy chitosan (NOCC); stem cells, islet of Langerhanscells and other cells for transplantation, including genetically alteredcells; red blood cells; white blood cells, including monocytes; andplatelets.

[0038] As used herein, the term “sterilize” is intended to mean areduction in the level of at least one active biological contaminant orpathogen found in the preparation containing a biological material beingtreated according to the present invention.

[0039] As used herein, the term “non-aqueous solvent” is intended tomean any liquid other than water in which a biological material may bedissolved or suspended or which may be disposed within a biologicalmaterial and includes both inorganic solvents and, more preferably,organic solvents. Illustrative examples of suitable non-aqueous solventsinclude, but are not limited to, the following: alkanes andcycloalkanes, such as pentane, 2-methylbutane (isopentane), heptane,hexane, cyclopentane and cyclohexane; alcohols, such as methanol,ethanol, 2-methoxyethanol, isopropanol, n-butanol, t-butyl alcohol, andoctanol; esters, such as ethyl acetate, 2-methoxyethyl acetate, butylacetate and benzyl benzoate; aromatics, such as benzene, toluene,pyridine, xylene; ethers, such as diethyl ether, 2-ethoxyethyl ether,ethylene glycol dimethyl ether and methyl t-butyl ether; aldehydes, suchas formaldehyde and glutaraldehyde; ketones, such as acetone and3-pentanone (diethyl ketone); glycols, including both monomeric glycols,such as ethylene glycol and propylene glycol, and polymeric glycols,such as polyethylene glycol (PEG) and polypropylene glycol (PPG), e.g.,PPG 400, PPG 1200 and PPG 2000; acids and acid anhydrides, such asformic acid, acetic acid, trifluoroacetic acid, phosphoric acid andacetic anhydride; oils, such as cottonseed oil, peanut oil, culturemedia, polyethylene glycol, poppyseed oil, safflower oil, sesame oil,soybean oil and vegetable oil; amines and amides, such as piperidine,N,N-dimethylacetamide and N,N-dimethylformamide; dimethylsulfoxide(DMSO); nitriles, such as benzonitrile and acetonitrile; hydrazine;detergents, such as polyoxyethylenesorbitan monolaurate (Tween 20) andmonooleate (Tween 80), Triton and sodium dodecyl sulfate; carbondisulfide; halogenated solvents, such as dichloromethane, chloroform,carbon tetrachloride, 1,2-dichlorobenzene, 1,2-dichloroethane,tetrachloroethylene and 1-chlorobutane; furans, such as tetrahydrofuran;oxanes, such as 1,4-dioxane; and glycerin/glycerol. Particularlypreferred examples of suitable non-aqueous solvents include non-aqueoussolvents which also function as stabilizers, such as ethanol andacetone.

[0040] As used herein, the term “biological contaminant or pathogen” isintended to mean a biological contaminant or pathogen that, upon director indirect contact with a biological material may have a deleteriouseffect on the biological material or upon a recipient thereof. Suchbiological contaminants or pathogens include the various viruses,bacteria, in both vegetative and spore states (including inter- andintracellular bacteria, such as mycoplasmas, ureaplasmas, nanobacteria,chlamydia, rickettsias), yeasts, molds, fungi, prions or similar agentsresponsible, alone or in combination, for TSEs and/or single ormulticellular parasites known to those of skill in the art to generallybe found in or infect biological materials. Examples of other biologicalcontaminants or pathogens include, but are not limited to, thefollowing: viruses, such as human immunodeficiency viruses and otherretroviruses, herpes viruses, filoviruses, circoviruses,paramyxoviruses, cytomegaloviruses, hepatitis viruses (includinghepatitis A, B, C, and D variants thereof, among others), pox viruses,toga viruses, Ebstein-Barr viruses and parvoviruses; bacteria, such asEscherichia, Bacillus, Campylobacter, Streptococcus and Staphylococcus;nanobacteria; parasites, such as Trypanosoma and malarial parasites,including Plasmodium species; yeasts; molds; fungi; mycoplasmas andureaplasmas; chlamydia; rickettsias, such as Coxiella bumetti; andprions and similar agents responsible, alone or in combination, for oneor more of the disease states known as transmissible spongiformencephalopathies (TSEs) in mammals, such as scrapie, transmissible minkencephalopathy, chronic wasting disease (generally observed in mule deerand elk), feline spongiform encephalopathy, bovine spongiformencephalopathy (mad cow disease), Creutzfeld-Jakob disease (includingvariant CJD), Fatal Familial Insomnia, Gerstmann-Straeussler-Scheinkersyndrome, kuru and Alpers syndrome. As used herein, the term “activebiological contaminant or pathogen” is intended to mean a biologicalcontaminant or pathogen that is capable of causing a deleterious effect,either alone or in combination with another factor, such as a secondbiological contaminant or pathogen or a native protein (wild-type ormutant) or antibody, in a biological material and/or a recipientthereof.

[0041] As used herein, the term “a biologically compatible solution” isintended to mean a solution to which a biological material may beexposed, such as by being suspended or dissolved therein, and retain itsessential biological and physiological characteristics. Such solutionsmay be of any suitable pH, tonicity, concentration and/or ionicstrength.

[0042] As used herein, the term “a biologically compatible bufferedsolution” is intended to mean a biologically compatible solution havinga pH and osmotic properties (e.g., tonicity, osmolality and/or oncoticpressure) suitable for maintaining the integrity of the material(s)therein. Suitable biologically compatible buffered solutions typicallyhave a pH between 2 and 8.5 and are isotonic or only moderatelyhypotonic or hypertonic. Biologically compatible buffered solutions areknown and readily available to those of skill in the art. Greater orlesser pH and/or tonicity may also be used in certain applications. Theionic strength of the solution may be high or low, but is typicallysimilar to the environments in which the tissue is intended to be used.

[0043] As used herein, the term “stabilizer” is intended to mean acompound or material that, alone and/or in combination, reduces damageto the biological material being irradiated to a level that isinsufficient to preclude the safe and effective use of the material.Illustrative examples of stabilizers that are suitable for use include,but are not limited to, the following, including structural analogs andderivatives thereof: antioxidants; free radical scavengers, includingspin traps, such as tert-butyl-nitrosobutane (tNB),α-phenyl-tert-butylnitrone (PBN), 5,5-dimethylpyrroline-N-oxide (DMPO),tert-butylnitrosobenzene (BNB), α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) and3,5-dibromo-4-nitroso-benzenesulphonic acid (DBNBS); combinationstabilizers, i.e., stabilizers which are effective at quenching bothType I and Type II photodynamic reactions; and ligands, ligand analogs,substrates, substrate analogs, modulators, modulator analogs,stereoisomers, inhibitors, and inhibitor analogs, such as heparin, thatstabilize the molecule(s) to which they bind. Preferred examples ofadditional stabilizers include, but are not limited to, the following:fatty acids, including 6,8-dimercapto-octanoic acid (lipoic acid) andits derivatives and analogues (alpha, beta, dihydro, bisnor and tetranorlipoic acid), thioctic acid, 6,8-dimercapto-octanoic acid,dihydrolopoate (DL-6,8-dithioloctanoic acid methyl ester), lipoamide,bisonor methyl ester and tetranor-dihydrolipoic acid, omega-3 fattyacids, omega-6 fatty acids, omega-9 fatty acids, furan fatty acids,oleic, linoleic, linolenic, arachidonic, eicosapentaenoic (EPA),docosahexaenoic (DHA), and palmitic acids and their salts andderivatives; carotenes, including alpha-, beta-, and gamma-carotenes;Co-Q10; xanthophylls; sucrose, polyhydric alcohols, such as glycerol,mannitol, inositol, and sorbitol; sugars, including derivatives andstereoisomers thereof, such as xylose, glucose, ribose, mannose,fructose, erythrose, threose, idose, arabinose, lyxose, galactose,allose, altrose, gulose, talose, and trehalose; amino acids andderivatives thereof, including both D- and L-forms and mixtures thereof,such as arginine, lysine, alanine, valine, leucine, isoleucine, proline,phenylalanine, glycine, serine, threonine, tyrosine, asparagine,glutamine, aspartic acid, histidine, N-acetylcysteine (NAC), glutamicacid, tryptophan, sodium capryl N-acetyl tryptophan, and methionine;azides, such as sodium azide; enzymes, such as Superoxide Dismutase(SOD), Catalase, and Δ4 , Δ5 and Δ6 desaturases; uric acid and itsderivatives, such as 1,3-dimethyluric acid and dimethylthiourea;allopurinol; thiols, such as glutathione and reduced glutathione andcysteine; trace elements, such as selenium, chromium, and boron;vitamins, including their precursors and derivatives, such as vitamin A,vitamin C (including its derivatives and salts such as sodium ascorbateand palmitoyl ascorbic acid) and vitamin E (and its derivatives andsalts such as alpha-, beta-, gamma-, delta-, epsilon-, zeta-, andeta-tocopherols, tocopherol acetate and alpha-tocotrienol);chromanol-alpha-C6; 6-hydroxy-2,5,7,8-tetramethylchroma-2 carboxylicacid (Trolox) and derivatives; extraneous proteins, such as gelatin andalbumin; tris-3-methyl-l-phenyl-2-pyrazolin-5-one (MCI-186); citiolone;puercetin; chrysin; dimethyl sulfoxide (DMSO); piperazinediethanesulfonic acid (PIPES); imidazole; methoxypsoralen (MOPS);1,2-dithiane-4, 5-diol; reducing substances, such as butylatedhydroxyanisole (BHA) and butylated hydroxytoluene (BHT); cholesterol,including derivatives and its various oxidized and reduced formsthereof, such as low density lipoprotein (LDL), high density lipoprotein(HDL), and very low density lipoprotein (VLDL); probucol; indolederivatives; thimerosal; lazaroid and tirilazad mesylate; proanthenols;proanthocyanidins; ammonium sulfate; Pegorgotein (PEG-SOD);N-tert-butyl-alpha-phenylnitrone (PBN);4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (Tempol); mixtures ofascorbate, urate and Trolox C (Asc/urate/Trolox C); proteins, such asalbumin, and peptides of two or more amino acids, any of which may beeither naturally occurring amino acids, i.e., L-amino acids, ornon-naturally occurring amino acids, i.e., D-amino acids, and mixtures,derivatives, and analogs thereof, including, but not limited to,arginine, lysine, alanine, valine, leucine, isoleucine, proline,phenylalanine, glycine, histidine, glutamic acid, tryptophan (Trp),serine, threonine, tyrosine, asparagine, glutamine, aspartic acid,cysteine, methionine, and derivatives thereof, such as N-acetylcysteine(NAC) and sodium capryl N-acetyl tryptophan, as well as homologousdipeptide stabilizers (composed of two identical amino acids), includingsuch naturally occurring amino acids, as Gly-Gly (glycylglycine) andTrp-Trp, and heterologous dipeptide stabilizers (composed of differentamino acids), such as carnosine (β-alanyl-histidine), anserine(β-alanyl-methylhistidine), and Gly-Trp; and flavonoids/flavonols, suchas diosmin, quercetin, rutin, silybin, silidianin, silicristin,silymarin, apigenin, apiin, chrysin, morin, isoflavone, flavoxate,gossypetin, myricetin, biacalein, kaempferol, curcumin, proanthocyanidinB2-3-O-gallate, epicatechin gallate, epigallocatechin gallate,epigallocatechin, gallic acid, epicatechin, dihydroquercetin, quercetinchalcone, 4,4′-dihydroxy-chalcone, isoliquiritigenin, phloretin,coumestrol, 4 ′, 7-dihydroxy-flavanone, 4 ′, 5-dihydroxy-flavone, 4 ′,6-dihydroxy-flavone, luteolin, galangin, equol, biochanin A, daidzein,formononetin, genistein, amentoflavone, bilobetin, taxifolin,delphinidin, malvidin, petunidin, pelargonidin, malonylapiin,pinosylvin, 3-methoxyapigenin, leucodelphinidin, dihydrokaempferol,apigenin 7-O-glucoside, pycnogenol, aminoflavone, purpurogallin fisetin,2 ′, 3′-dihydroxyflavone, 3-hydroxyflavone, 3 ′, 4′-dihydroxyflavone,catechin, 7-flavonoxyacetic acid ethyl ester, catechin, hesperidin, andnaringin. Particularly preferred examples include single stabilizers orcombinations of stabilizers that are effective at quenching both Type Iand Type II photodynamic reactions, and volatile stabilizers, which canbe applied as a gas and/or easily removed by evaporation, low pressure,and similar methods. Additional preferred examples for use in themethods of the present invention include hydrophobic stabilizers.

[0044] As used herein, the term “residual solvent content” is intendedto mean the amount or proportion of freely-available liquid in thebiological material. Freely-available liquid means the liquid, such aswater and/or an organic solvent (e.g., ethanol, isopropanol,polyethylene glycol, etc.), present in the biological material beingsterilized that is not bound to or complexed with one or more of thenon-liquid components of the biological material. Freely-availableliquid includes intracellular water and/or other solvents. The residualsolvent contents related as water referenced herein refer to levelsdetermined by the FDA approved, modified Karl Fischer method (Meyer andBoyd, Analytical Chem., 31:215-219, 1959; May, et al., J. Biol.Standardization, 10:249-259, 1982; Centers for Biologics Evaluation andResearch, FDA, Docket No. 89D-0140, 83-93; 1990) or by near infraredspectroscopy. Quantitation of the residual levels of water or othersolvents may be determined by means well known in the art, dependingupon which solvent is employed. The proportion of residual solvent tosolute may also be considered to be a reflection of the concentration ofthe solute within the solvent. When so expressed, the greater theconcentration of the solute, the lower the amount of residual solvent.

[0045] As used herein, the term “sensitizer” is intended to mean asubstance that selectively targets viruses, bacteria, in both vegetativeand spore states (including inter- and intracellular bacteria, such asmycoplasmas, ureaplasmas, nanobacteria, chlamydia, rickettsias), yeasts,molds, fungi, single or multicellular parasites, and/or prions orsimilar agents responsible, alone or in combination, for TSEs, renderingthem more sensitive to inactivation by radiation, therefore permittingthe use of a lower rate or dose of radiation and/or a shorter time ofirradiation than in the absence of the sensitizer. Illustrative examplesof suitable sensitizers include, but are not limited to, the following:psoralen and its derivatives and analogs (including 3-carboethoxypsoralens); inactines and their derivatives and analogs; angelicins,khellins and coumarins which contain a halogen substituent and a watersolubilization moiety, such as quaternary ammoniumion or phosphoniumion; nucleic acid binding compounds; brominated hematoporphyrin;phthalocyanines; purpurins; porphyrins; halogenated or metalatom-substituted derivatives of dihematoporphyrin esters,hematoporphyrin derivatives, benzoporphyrin derivatives,hydrodibenzoporphyrin dimaleimade, hydrodibenzoporphyrin, dicyanodisulfone, tetracarbethoxy hydrodibenzoporphyrin, and tetracarbethoxyhydrodibenzoporphyrin dipropionamide; doxorubicin and daunomycin, whichmay be modified with halogens or metal atoms; netropsin; BD peptide, S2peptide; S-303 (ALE compound); dyes, such as hypericin, methylene blue,eosin, fluoresceins (and their derivatives), flavins, merocyanine 540;photoactive compounds, such as bergapten; and SE peptide. In addition,atoms which bind to prions, and thereby increase their sensitivity toinactivation by radiation, may also be used. An illustrative example ofsuch an atom would be the Copper ion, which binds to the prion proteinand, with a Z number higher than the other atoms in the protein,increases the probability that the prion protein will absorb energyduring irradiation, particularly gamma irradiation.

[0046] As used herein, the term “radiation” is intended to meanradiation of sufficient energy to sterilize at least some component ofthe irradiated biological material. Types of radiation include, but arenot limited to, the following: (i) corpuscular (streams of subatomicparticles such as neutrons, electrons, and/or protons); (ii)electromagnetic (originating in a varying electromagnetic field, such asradio waves, visible (both mono and polychromatic) and invisible light,infrared, ultraviolet radiation, x-radiation, and gamma rays andmixtures thereof); and (iii) sound and pressure waves. Such radiation isoften described as either ionizing (capable of producing ions inirradiated materials) radiation, such as gamma rays, and non-ionizingradiation, such as visible light. The sources of such radiation may varyand, in general, the selection of a specific source of radiation is notcritical provided that sufficient radiation is given in an appropriatetime and at an appropriate rate to effect sterilization. In practice,gamma radiation is usually produced by isotopes of Cobalt or Cesium,while UV and X-rays are produced by machines that emit UV andX-radiation, respectively, and electrons are often used to sterilizematerials in a method known as “E-beam” irradiation that involves theirproduction via a machine. Visible light, both mono- and polychromatic,is produced by machines and may, in practice, be combined with invisiblelight, such as infrared and UV, that is produced by the same machine ora different machine.

[0047] As used herein, the term “tissue” is intended to mean a substancederived or obtained from a multi-cellular living organism that performsone or more functions in the organism or a recipient thereof. Thus, asused herein, a “tissue” may be an aggregation of intercellularsubstance(s), such as collagen, elastin, fibronectin, fibrin,glycosaminoglycans and the like, and/or cells which are generallymorphologically similar, such as hemapoietic cells, bone cells and thelike. Accordingly, the term “tissue” is intended to include bothallogenic and autologous tissue, including, but not limited to, cellularviable tissue, cellular non-viable tissue and acellular tissue, such ascollagen, elastin, fibronectin, fibrin, glycosaminoglycans and the like.As used herein, the term “tissue” includes naturally occurring tissues,such as tissues removed from a living organism and used as such, orprocessed tissues, such as tissue processed so as to be less antigenic,for example allogenic tissue intended for transplantation, and tissueprocessed to allow cells to proliferate into the tissue, for exampledemineralised bone matrix that has been processed to enable bone cellsto proliferate into and through it or heart valves that have beenprocessed to encourage cell engraftment following implantation.Additionally, as used herein, the term “tissue” is intended to includenatural, artificial, synthetic, semi-synthetic or semi-artificialmaterials comprised of biomolecules structured in such a way as toperrnit the replacement of at least some function(s) of a natural tissuewhen implanted into a recipient. Such constructs may be placed in acell-containing environment prior to implantation to encourage theircellularization. Illustrative examples of tissues that may be treatedaccording to the methods of the present invention include, but are notlimited to, the following: connective tissue; epithelial tissue; adiposet issue; cartilage, bone (including d emineralised bone matrix);muscle tissue; and nervous tissue. Non-limiting examples of specifictissues that may be treated according to the methods of the presentinvention include heart, lung, liver, spleen, pancreas, kidney, comeas,joints, bone marrow, blood cells (red blood cells, leucocytes,lymphocytes, platelets, etc.), plasma, skin, fat, tendons, ligaments,hair, muscles, blood vessels (arteries, veins), teeth, gum tissue,fetuses, eggs (fertilized and not fertilized), eye lenses, hands, nervecells, nerves, and other physiologically and anatomically complextissues, such as intestine, cartilage, entire limbs, cadavers, andportions of brain, and intracellular substances, such as collagen,elastin, fibrinogen, fibrin, fibronectin, glycosaminoglycans, andpolysaccharides.

[0048] As used herein, the term “to protect” is intended to mean toreduce any damage to the biological material being irradiated, thatwould otherwise result from the irradiation of that material, to a levelthat is insufficient to preclude the safe and effective use of thematerial following irradiation. In other words, as ubstance or process“protects” a biological material from radiation if the presence of thatsubstance or carrying out that process results in less damage to thematerial from irradiation than in the absence of that substance orprocess. Thus, a biological material may be used safely and effectivelyafter irradiation in the presence of a substance or followingperformance of a process that “protects” the material, but could not beused with as great a degree of safety or as effectively afterirradiation under identical conditions but in the absence of thatsubstance or the performance of that process.

[0049] As used herein, an “acceptable level” of damage may varydepending upon certain features of the particular method(s) of thepresent invention being employed, such as the nature and characteristicsof the particular biological material and/or non-aqueous solvent(s)being used, and/or the intended use of the material being irradiated,and can be determined empirically by one skilled in the art. An“unacceptable level” of damage would therefore be a level of damage thatwould preclude the safe and effective use of the biological materialbeing sterilized. The particular level of damage in a given biologicalmaterial may be determined using any of the methods and techniques knownto one skilled in the art.

B. Particularly Preferred Embodiments

[0050] A first particularly preferred embodiment of the presentinvention is directed to a device for measuring the amount of energyabsorbed by a product undergoing sterilization with radiation,comprising: (i) an effective amount of at least one material thatabsorbs radiation in a quantifiable manner; and (ii) an effective amountof at least one cooling agent for maintaining said material within apredetermined temperature range between −120° C. and ambient temperatureduring irradiation.

[0051] Suitable materials that absorb radiation in a quantifiable mannerare known and available to those skilled in the art. Preferred materialsfor use in the devices of the present invention include materials which,when irradiated, exhibit a reproducible change in some quantifiableproperty, e.g, a physical or chemical property of the material, that isrelated to the amount of radiation absorbed by the material. Forexample, suitable materials may exhibit changes in absorption spectra,luminescence, or free radical generation.

[0052] The change in the quantifiable property of the material may bemeasured using any of the methods and techniques known to those skilledin the art, such as spectrophotometry, spectrometry and micrometry.Illustrative examples of suitable techniques for use with the devices ofthe present invention include, but are not limited to, electron spinresonance (ESR) spectroscopy, UV spectrophotometry, electrochemicalpotentiation, color titration, UV/VIS spectrophotometry and colorimetry.

[0053] Illustrative examples of suitable materials that may be used inthe devices of the present invention include, but are not limited to,alanine, cellulose acetate, ceric/cerous sulfate, potassium/silverdichromate, ferrous sulfate, radiochromic dye solutions, ethanolchlorobenzene, triphenyl methyl cyanide, crystalline solids such asalkali halides (e.g., LiF and the like) and radiochromic films.

[0054] In particularly preferred embodiments of the present invention,the material that absorbs radiation in a quantifiable manner is selectedfrom among the following: alanine, cellulose acetate, ethanolchlorobenzene and radiochromic films.

[0055] The material which absorbs radiation in a quantifiable manner maybe employed in the inventive device alone, for example, as pellets (suchas alanine pellets). Alternatively, a plurality of different materialswhich absorb radiation in a quantifiable manner may be employed incombination. Suitable a mounts of such material(s) may be determinedempirically by one skilled in the art.

[0056] According to still other preferred embodiments of the presentinvention, the material (or materials) that absorbs radiation may bemixed with one or more other suitable ingredients, such as binders andthe like. The relative amounts of the material which absorbs radiationand any other ingredients that may be present, such as a binder, may bedetermined empirically by one skilled in the art using methods andtechniques known in the art.

[0057] The material(s) that absorbs radiation in a quantifiable manner,and any other ingredients that may be present, may be formulated usingany of the methods and materials known and available to those skilled inthe art. For example, the material(s), and any other ingredient(s), maybe molded, extruded or otherwise shaped or formed into a shape suitablefor use in the devices of the present invention.

[0058] Illustrative examples of suitable binders that may be employed indevices according to certain preferred embodiments of the presentinvention include, but are not limited to, natural rubber, syntheticrubber and mixtures thereof. Illustrative examples of suitable syntheticrubbers include, but are not limited to, ethylene propylene copolymer,ethylene-vinyl acetate copolymer, chloroprene rubber, nitrile rubber,butyl rubber, synthetic isoprene rubber, styrene-butadiene copolymer,styrene-butadiene-acrylonitrile copolymer, butadiene rubber, acrylicrubber, urethane rubber, silicone rubber, polyisobutylene, polyesterrubber, epichlorohydrin rubber and tetrafluoroethylene-propylenealternating copolymer.

[0059] Still other illustrative examples of suitable binders includepolymers, such as ethylene-propylene copolymers, polyethylene, includinglow density polyethylene and very low density polyethylene,polyisobutylene, polyethylene terephthalates, polyamides, ethylene-vinylacetate copolymers, polypropylene, polymethylmethacrylate (PMMA), methylpentene, polycarbonate, polystyrene thermoset polymers, fluoropolymers,such as teflon and the like. Mixtures of such polymers may also beemployed as the binder in the devices of the present invention.

[0060] Suitable cooling agents that may be employed in the devices ofthe present invention include any material capable of maintaining thematerial that absorbs radiation in a quanifiable manner within thepredetermined temperature range. Such materials may be a solid orsemi-solid, and may be in the form of particles, sheets, pellets and thelike.

[0061] Illustrative examples of suitable cooling agents include, but arenot limited, the following: dry ice, water ice, combinations of dry iceand non-aqueous solvents (also know as “dry ice baths”), such asmethanol, ethanol, isopropanol, ethylene glycol (alone or thinned withwater or isopropanol), acetone, dichloromethane, chloroform, carbontetrachloride and the like. Such cooling agents may be used alone or incombination.

[0062] In certain particularly preferred embodiments of the presentinvention, the cooling agent is dry ice. According to these embodimentsof the present invention, the cooling agent is preferably in the form ofparticles. Preferably, the particles of cooling agent have an averagevolume of not more than 17 cm³ (about 1 in³). More preferably, theparticles of cooling agent have an average volume of not more than 1 cm³and even more preferably not more than 0.5 cm³.

[0063] According to certain preferred embodi agent is of sufficientvolume to contain at least a portion of the material that absorbsradiation. For example, in a particularly preferred embodiment of thepresent, the cooling agent may be in the form of a block of dry ice withan opening sufficiently large to hold at least a portion of the materialthat absorbs radiation. Such an opening, e.g. a hole or depression inthe block, may be drilled or otherwise prepared and then at least aportion of the material that absorbs radiation may be placed in thatopening. Such openings may have any shape suitable to hold at least aportion of the material that absorbs radiation, such as rectangular,circular, square and the like. Preferably, the opening is of asufficient size to contain substantially all of the material thatabsorbs radiation.

[0064] According to still other preferred embodiments of the presentinvention, the device for measuring the amount of energy absorbed by aproduct undergoing irradiation also includes a container of sufficientvolume to contain at least a portion of the cooling agent and at least aportion of the material that absorbs radiation. Preferably, such acontainer is of sufficient volume to contain at least a portion of thecooling agent and substantially all of the material that absorbsradiation. Suitable containers include, but are not limited to, vacuumDewars and other insulating containers, such as, polystyrene containersand the like.

[0065] In accordance with the various embodiments of the presentinvention, the cooling agent is capable of maintaining the material thatabsorbs energy in a quantifiable manner within a predetenninedtemperature range during irradiation of the material. Preferably, thetemperature range is between about −120° C. and ambient temperature.

[0066] A second particularly preferred embodiment of the presentinvention is directed to a method for determining the amount of energyabsorbed by a product undergoing sterilization with radiation. Accordingto such preferred embodiments of the present invention, such methodsinclude placing within a suitable container at least one product to besterilized and at least one device, wherein the device comprises atleast one material that absorbs radiation in a quantifiable manner andan effective amount of at least one cooling agent for maintaining thematerial within a predetermined temperature range, preferably betweenabout −120° C. and ambient temperature, during irradiation; irradiatingthe container containing the product and the device; and analyzing thematerial that absorbs energy to determine the amount of energy absorbedduring irradiation.

[0067] A third particularly preferred embodiment of the presentinvention is directed to a method for maintaining the temperature of aproduct undergoing irradiation within a predetermined temperature range,preferably between about −120° C. and ambient temperature. According tosuch preferred embodiments of the present invention, the method includesplacing at least one product to be irradiated in a suitable containerhaving at least one side and a bottom, wherein the volume defined by thecontainer is greater than the volume of the product; placing aneffective amount of at least one cooling agent in the container betweenthe product and the at least one side; and irradiating the containercontaining the product and the cooling agent with ionizing radiation.

[0068] According to certain preferred embodiments of the presentinvention, the product being irradiated may be a biological material.According to other preferred embodiments of the present invention, theproduct may be a material that absorbs radiation in a quantifiablemanner. In either such embodiment, the product is preferably frozen.

[0069] According to certain preferred embodiments of the presentinvention, the product may be a biological material selected from thegroup consisting of dextrose; urokinase; thrombin; trypsin; antithrombinIII; plasminogen; plasma; purified protein fraction; blood; blood cells;alpha 1 proteinase inhibitor; digestive enzymes, such as galactosidasesand sulfatases.; blood proteins, such as albumin, Factor VIII, FactorVII, Factor IV, fibrinogen, monoclonal immunoglobulins and polyclonalimmunoglobulins; and tissue, such as heart valves, ligaments,demineralized bone matrix, tendons, nerves, bone, teeth, bone marrow,skin grafts, cartilage, corneas, arteries, veins and organs fortransplantation.

[0070] According to certain preferred embodiments of the presentinvention, the container having at least one side and a bottom may be avacuum Dewar or a foam box, such as a polystyrene foam box.

[0071] In still other preferred embodiments of the present invention,this container having at least one side and a bottom may include a frontside and a back side and a first side and a second side. According tosuch embodiments, the cooling agent is preferably placed between theproduct being irradiated and the first side and/or between the productbeing irradiated and the second side.

[0072] According to the methods of the present invention, any suitableradiation dose may be employed. According to preferred embodiments, suchas when the product includes a biological material, the radiation doseis sufficient for sterilization.

[0073] According to the methods of the present invention, thepredetermined temperature range is within the range of −120° C. toambient temperature. In preferred embodiments of the present invention,both endpoints of the predetermined temperature range are less thanambient temperature.

[0074] According to other preferred embodiments of the presentinvention, at least one of the endpoints of the predeterminedtemperature range is less than the freezing point of the product beingirradiated. More preferably, both endpoints of the temperature range areless than the freezing point of the product being irradiated.

[0075] According to still other preferred embodiments of the presentinvention, at least one endpoint of the predetermined temperature rangeis less than the glass transition temperature of the product beingirradiated. In still other preferred embodiments of the presentinvention, both endpoints of the temperature range are less than theglass transition temperature of the product.

[0076] In certain preferred embodiments of the present invention, atleast one of the endpoints of the predetermined temperature range ispreferably less than −20° C., more at least one endpoint is preferablyless than about −40° C., still more preferably at least one endpoint isless than about −60° C., and most preferably at least one endpoint isless than about −70° C.

[0077] In still other preferred embodiments of the present invention,both endpoints of the predetermined temperature range are less thanabout −20° C., more preferably both endpoints are less than about −40°C., still more preferably both endpoints are less than about −60° C.,and most preferably both endpoints are less than about −70° C.

[0078] In certain other preferred embodiments of the present invention,the predetermined temperature range is less than the increase intemperature that would occur under adiabatic conditions.

[0079] According to other preferred embodiments of the presentinvention, the predetermined temperature range is less than 10° C. Morepreferably, the predetermined temperature range is less than 5° C.,still more preferably the predetermined temperature range is less thanabout 2° C., even more preferably the predetermined temperature range isless than about 1.25° C., still even more preferably the predeterminedtemperature range is less than about 0.65° C., yet even still morepreferably the predetermined temperature range is less than about 0.25°C., and most preferably the predetermined temperature range is less thanabout 0.1° C.

[0080] According to certain other preferred embodiments of the presentinvention, the predetermined temperature range is less than 0.1° C. perkGy (kiloGray) of radiation. More preferably, the predeterminedtemperature range is less than 0.05° C. per kGy of radiation, still morepreferably the predetermined temperature range is less than about 0.02°C. per kGy of radiation, even more preferably the predeterminedtemperature range is less than about 0.0125° C. per kGy of radiation,and most preferably the predetermined temperature range is less thanabout 0.0065° C. per kGy of radiation.

[0081] The devices and methods of the present invention are particularlyuseful when employed in sterilization processes involving ionizingradiation, such as gamma radiation. For example, the devices and methodsof the present invention may be used in conjunction with processes forsterilizing a preparation containing a biological material by subjectingthat preparation to an effective amount of gamma radiation.

[0082] According to such preferred embodiments of the present invention,the preparation containing a biological material to be irradiated may besubjected to at least one, more preferably at least two, stabilizingprocesses prior to sterilization. Such stabilizing processes include,but are not limited to, adding to the preparation containing abiological material at least one stabilizer, reducing the residualsolvent content of the preparation containing a biological material,reducing the temperature of the preparation containing a biologicalmaterial, reducing the oxygen content of the preparation containing abiological material, maintaining or adjusting the pH of the preparationcontaining a biological material and adding to the preparationcontaining a biological material at least one non-aqueous solvent.

[0083] The preparation containing a biological material may contain amixture of water and a non-aqueous solvent, such as ethanol and/oracetone. In such embodiments, the non-aqueous solvent(s) is (are)preferably a non-aqueous solvent that is not prone to the formation offree-radicals upon irradiation, and most preferably a non-aqueoussolvent that is not prone to the formation of free-radicals uponirradiation and that has little or no dissolved oxygen or other gas(es)that is (are) prone to the formation of free-radicals upon irradiation.Volatile non-aqueous solvents are particularly preferred, even moreparticularly preferred are non-aqueous solvents that are alsostabilizers, such as ethanol and acetone.

[0084] According to certain methods, a stabilizer is added prior toirradiation of the preparation containing a biological material withradiation. This stabilizer is preferably added to the preparationcontaining a biological material in an amount that is effective toprotect the preparation containing a biological material from theradiation. Alternatively, the stabilizer is added to the preparationcontaining a biological material in an amount that, together with anon-aqueous solvent, is effective to protect the preparation containinga biological material from the radiation. Suitable amounts of stabilizermay vary depending upon certain features of the particular method(s) ofthe present invention being employed, such as the particular stabilizerbeing used and/or the nature and characteristics of the particularpreparation containing a biological material being irradiated and/or itsintended use, and can be determined empirically by one skilled in theart.

[0085] According to certain methods, the residual solvent content of thepreparation containing a biological material is reduced prior toirradiation. The residual solvent content is preferably reduced to alevel that is effective to protect the preparation containing abiological material from the radiation. Suitable levels of residualsolvent content may vary depending upon certain features of theparticular method(s) of the present invention being employed, such asthe nature and characteristics of the particular preparation containinga biological material being irradiated and/or its intended use, and canbe determined empirically by one skilled in the art. There may bepreparations containing biological materials for which it is desirableto maintain the residual solvent content to within a particular range,rather than a specific value.

[0086] According to certain methods, when the preparation containing abiological material also contains water, the residual solvent (water)content of the preparation containing a biological material may bereduced by dissolving or suspending the preparation containing abiological material in a non-aqueous solvent that is capable ofdissolving water. Preferably, such a non-aqueous solvent is not prone tothe formation of free-radicals upon irradiation and has little or nodissolved oxygen or other gas(es) that is (are) prone to the formationof free-radicals upon irradiation.

[0087] While not wishing to be bound by any theory of operability, it isbelieved that the reduction in residual solvent content reduces thedegrees of freedom of the preparation containing a biological material,reduces the number of targets for free radical generation and mayrestrict the diffusability of these free radicals. Similar results mighttherefore be achieved by lowering the temperature of the preparationscontaining a biological material below their eutectic point(s) or belowtheir freezing point(s), or by vitrification to likewise reduce thedegrees of freedom of the preparation containing a biological material.These results may permit the use of a higher rate and/or dose ofradiation than might otherwise be acceptable. Thus, the methodsdescribed herein may be performed at any temperature that doesn't resultin unacceptable damage to the preparation containing a biologicalmaterial, i.e., damage that would preclude the safe and effective use ofthe preparation containing a biological material. Preferably, themethods described herein are performed at ambient temperature or belowambient temperature, such as below the eutectic point(s) or freezingpoint(s) of the preparation containing a biological material beingirradiated.

[0088] In certain embodiments, the desired residual solvent content of aparticular preparation containing a biological material may be found tolie within a range, rather than at a specific point. Such a range forthe preferred residual solvent content of a particular biologicalmaterial may be determined empirically by one skilled in the art.

[0089] The residual solvent content of the preparation containing abiological material may be reduced by any of the methods and techniquesknown to those skilled in the art for reducing solvent from thepreparation containing a biological material without producing anunacceptable level of damage to the preparation containing a biologicalmaterial. Such methods include, but are not limited to, lyophilization,drying, concentration, addition of alternative solvents, evaporation,chemical extraction and vitrification.

[0090] A particularly preferred method for reducing the residual solventcontent of a preparation containing a biological material islyophilization.

[0091] Another particularly preferred method for reducing the residualsolvent content of a preparation containing a biological material isvitrification, which may be accomplished by any of the methods andtechniques known to those skilled in the art, including the addition ofsolute and or additional solutes, such as sucrose, to raise the eutecticpoint(s) of the preparation containing a biological material, followedby a gradual application of reduced pressure to the preparationcontaining a biological material in order to remove the residualsolvent. The resulting glassy material will then have a reduced residualsolvent content.

[0092] According to certain methods, the preparation containing abiological material to be sterilized may be immobilized upon or attachedto a solid surface by any means known and available to one skilled inthe art. For example, the preparation containing a biological materialto be sterilized may be attached to a biological or non-biologicalsubstrate.

[0093] In another preferred embodiment, where the preparation containinga biological material contains oxygen or other gases dissolved withinthe preparation containing a biological material or within theircontainer or associated with them, the amount of these gases within orassociated with the preparation containing a biological material may bereduced by any of the methods and techniques known and available tothose skilled in the art, such as the controlled reduction of pressurewithin a container (rigid or flexible) holding the preparationcontaining a biological material to be treated or by placing thepreparation containing a biological material in a container ofapproximately equal volume.

[0094] In certain embodiments, when the preparation containing abiological material to be treated contains an aqueous or non-aqueoussolvent, or a mixture of such solvents, at least one stabilizer isintroduced according to any of the methods and techniques known andavailable to one skilled in the art, including soaking the tissue in asolution containing the stabilizer(s), preferably under pressure, atelevated temperature and/or in the presence of a penetration enhancer,such as dimethylsulfoxide, and more preferably, when the stabilizer(s)is a protein, at a high concentration. Other methods of introducing atleast one stabilizer into tissue include, but are not limited to, thefollowing: applying a gas containing the stabilizer(s), preferably underpressure and/or at elevated temperature; injecting the stabilizer(s) ora solution containing the stabilizer(s) directly into the tissue;placing the tissue under reduced pressure and then introducing a gas orsolution containing the stabilizer(s); dehydrating the tissue, such asby using a buffer of high ionic and/or osmolar strength, and rehydratingthe tissue with a solution containing the stabilizer(s); applying a highionic strength solvent c ontaining the stabilizer(s), which mayoptionally be followed by a controlled reduction in the ionic strengthof the solvent; cycling the tissue between solutions of high ionicand/or osmolar strength and solutions of low ionic and/or osmolarstrength containing the stabilizer(s); and combinations of two or moreof these methods. One or more sensitizers may also be introduced intotissue according to such methods.

[0095] According to certain embodiments, in order to enhance penetrationof one or more stabilizers and/or sensitizers into the tissue, one ormore compounds effective to increase penetration into the tissue may beemployed. For instance, the tissue may treated with one or morecompounds that cause an increase in the distance between molecules inthe tissue, thereby promoting penetration of the stabilizers and/orsensitizers into the tissue.

[0096] Similarly, the tissue may be treated with one or more compoundsthat cause macromolecules in the tissue to become less compact, orrelaxed, thereby promoting penetration of the stabilizer(s) and/orsensitizer(s) into the tissue or providing a greater surface area oftissue to be in contact with the stabilizer(s) and/or sensitizer(s). Thecompounds that cause macromolecules in the tissue to become lesscompact, or relaxed, may also be applied prior to introduction of thestabilizer(s) and/or sensitizer(s), which may then be introduced in asimilar solution followed by application of a solution containing asimilar amount of stabilizer(s) and/or sensitizer(s) but a reducedamount of the compounds that cause macromolecules in the tissue tobecome less compact, or relaxed. Repeated applications of suchsolutions, with progressively lower amounts of compounds that causemacromolecules in the tissue to become less compact, or relaxed, maysubsequently be applied.

[0097] The compounds that promote penetration may be used alone or incombination, such as a combination of a compound that causesmacromolecules in the tissue to become less compact and a compound thatcauses an increase in the distance between molecules in the tissue.

[0098] Further, in those embodiments wherein the stabilizer(s) and/orsensitizer(s) is cationic, one or more anionic compounds may be added tothe solution containing the stabilizer(s) and/or sensitizer(s) prior toand/or during application thereof to the preparation containing abiological material. The anionic compound(s) may also be applied priorto introduction of the stabilizer(s) and/or sensitizer(s), which maythen be introduced in a similar solution followed by application of asolution containing a similar amount of stabilizer(s) and/orsensitizer(s) but a reduced amount of the anionic compound(s). Repeatedapplications of such solutions, with progressively lower amounts ofanionic compound(s) may subsequently be applied.

[0099] Similarly, in those embodiments wherein the stabilizer(s) and/orsensitizer(s) is anionic, one or more cationic compounds may be added tothe solution containing the stabilizer(s) and/or sensitizer(s) prior toand/or during application thereof to the preparation containing abiological material. The cationic compound(s) may also be applied priorto introduction of the stabilizer(s) and/or sensitizer(s), which maythen be introduced in a similar solution followed by application of asolution containing a similar amount of stabilizer(s) and/orsensitizer(s) but a reduced amount of the cationic compound(s). Repeatedapplications of such solutions, with progressively lower amounts ofcationic compound(s) may subsequently be applied.

[0100] It will be appreciated that the combination of one or more of thefeatures described herein may be employed to further minimizeundesirable effects upon the preparation containing a biologicalmaterial caused by irradiation, while maintaining adequate effectivenessof the irradiation process on the biological contaminant(s) orpathogen(s). For example, in addition to the use of a stabilizer, aparticular preparation containing a biological material may also belyophilized, held at a reduced temperature and kept under vacuum priorto irradiation to further minimize undesirable effects.

[0101] The radiation employed may be any radiation effective for thesterilization of the preparation containing a biological material beingtreated. The radiation may be corpuscular, including E-beam radiation.Preferably the radiation is electromagnetic radiation, including x-rays,infrared, visible light, UV light and mixtures of various wavelengths ofelectromagnetic radiation. A particularly preferred form of radiation isgamma radiation.

[0102] According to these preferred methods, the preparation containinga biological material is irradiated with the radiation at a rateeffective for the sterilization of the preparation containing abiological material, while not producing an unacceptable level of damageto the preparation containing a biological material. Suitable rates ofirradiation may vary depending upon certain features of the methods ofthe present invention being employed, such as the nature andcharacteristics of the particular preparation containing a biologicalmaterial, which may contain a non-aqueous solvent, being irradiated, theparticular form of radiation involved, and/or the particular biologicalcontaminants or pathogens being inactivated. Suitable rates ofirradiation can be determined empirically by one skilled in the art.Preferably, the rate of irradiation is constant for the duration of thesterilization procedure. When this is impractical or otherwise notdesired, a variable or discontinuous irradiation may be utilized.

[0103] According to these methods, the rate of irradiation may beoptimized to produce the most advantageous combination of productrecovery and time required to complete the operation. Both low (<3kGy/hour) and high (>3 kGy/hour) rates may be utilized in the methodsdescribed herein to achieve such results. The rate of irradiation ispreferably selected to optimize the recovery of the preparationcontaining a biological material while still sterilizing the preparationcontaining a biological material. Although reducing the rate ofirradiation may serve to decrease damage to the preparation containing abiological material, it will also result in longer irradiation timesbeing required to achieve a particular desired total dose. A higher doserate may therefore be preferred in certain circumstances, such as tominimize logistical issues and costs, and may be possible particularlywhen used in accordance with the methods described herein for protectingthe preparation containing a biological material from irradiation.

[0104] According to particularly preferred methods, the rate ofirradiation is not more than about 3.0 kGy/hour, more preferably betweenabout 0.1 kGy/hr and 3.0 kGy/hr, even more preferably between about 0.25kGy/hr and 2.0 kGy/hour, still even more preferably between about 0.5kGy/hr and 1.5 kGy/hr and most preferably between about 0.5 kGy/hr and1.0 kGy/hr.

[0105] According to other particularly preferred methods, the rate ofirradiation is at least about 3.0 kGy/hr, more preferably at least about6 kGy/hr, even more preferably at least about 16 kGy/hr, even morepreferably at 1 east a bout 30 k Gy/hr and m ost preferably at 1 east about 45 kGy/hr or greater.

[0106] According to these methods, the preparation containing abiological material to be sterilized may be irradiated with theradiation for a time effective for the sterilization of the preparationcontaining a biological material. Combined with irradiation rate, theappropriate irradiation time results in the appropriate dose ofirradiation being applied to the preparation containing a biologicalmaterial. Suitable irradiation times may vary depending upon theparticular form and rate of radiation involved and/or the nature andcharacteristics of the particular preparation containing a biologicalmaterial being irradiated. Suitable irradiation times can be determinedempirically by one skilled in the art.

[0107] According to these methods, the preparation containing abiological material to be sterilized is irradiated with radiation up toa total dose effective for the sterilization of the preparationcontaining a biological material, while not producing an unacceptablelevel of damage to those preparations containing a biological material.Suitable total doses of radiation may vary depending upon certainfeatures of the methods of the present invention being employed, such asthe nature and characteristics of the particular preparation containinga biological material being irradiated, the particular form of radiationinvolved, and/or the particular biological contaminants or pathogensbeing inactivated. Suitable total doses of radiation can be determinedempirically by one skilled in the art. Preferably, the total dose ofradiation is at least 25 kGy, more preferably at least 45 kGy, even morepreferably at least 75 kGy, and still more preferably at least 100 kGyor greater, such as 150 kGy or 200 kGy or greater.

[0108] The particular geometry of the preparation containing abiological material being irradiated, such as the thickness and distancefrom the source of radiation, may be determined empirically by oneskilled in the art. A preferred embodiment is a geometry that providesfor an even rate of irradiation throughout the preparation containing abiological material. A particularly preferred embodiment is a geometrythat results in a short path length for the radiation through thepreparation, thus minimizing the differences in radiation dose betweenthe front and back of the preparation. This may be further minimized insome preferred geometries, particularly those wherein the preparationcontaining a biological material has a relatively constant radius aboutits axis that is perpendicular to the radiation source and by theutilization of a means of rotating the preparation containing abiological material about said axis.

[0109] Similarly, according to certain methods, an effective package forcontaining the preparation containing a biological material duringirradiation is one which combines stability under the influence ofirradiation, and which minimizes the interactions between the package ofthe preparation containing a biological material and the radiation.Preferred packages maintain a seal against the external environmentbefore, during and post-irradiation, and are not reactive with thepreparation containing a biological material within, nor do they producechemicals that may interact with the preparation containing a biologicalmaterial within. Particularly preferred examples include but are notlimited to containers that comprise glasses stable when irradiated,stoppered with stoppers made of rubber or other suitable materials thatis relatively stable during radiation and liberates a minimal amount ofcompounds from within, and sealed with metal crimp sales of aluminum orother suitable materials with relatively low Z numbers. S uitablematerials can be determined by measuring their physical performance, andthe amount and type of reactive leachable compounds post-irradiation,and by examining other characteristics known to be important to thecontainment of such biological materials as preparation containing abiological material empirically by one skilled in the art.

[0110] According to certain methods, an effective amount of at least onesensitizing compound may optionally be added to the preparationcontaining a biological material prior to irradiation, for example toenhance the effect of the irradiation on the biological contaminant(s)or pathogen(s) therein, while employing the methods described herein tominimize the deleterious effects of irradiation upon the preparationcontaining a biological material. Suitable sensitizers are known tothose skilled in the art, and include psoralens and their derivativesand inactines and their derivatives.

[0111] According to these methods, the irradiation of the preparationcontaining a biological material may occur at any temperature belowambient, such as 0° C., −20° C., −40° C., −60° C., −70° C. or −78° C.According to this embodiment, the preparation containing a biologicalmaterial is preferably irradiated at or below the freezing or eutecticpoint(s) of the preparation containing a biological material or theresidual solvent therein. Most preferably, the irradiation of thepreparation containing a biological material occurs at a temperaturethat protects the preparation containing a biological material fromradiation. Suitable temperatures can be determined empirically by oneskilled in the art.

[0112] In certain embodiments, the temperature at which irradiation isperformed may be found to lie within a range, rather than at a specificpoint. Such a range for the preferred temperature for the irradiation ofa particular preparation containing a biological material may bedetermined empirically by one skilled in the art.

[0113] According to these methods, the irradiation of the preparationcontaining a biological material may occur at any pressure which is notdeleterious to the preparation containing a biological material beingsterilized. According to one preferred embodiment, the preparationcontaining a biological material is irradiated at elevated pressure.More preferably, the preparation containing a biological material isirradiated at elevated pressure due to the application of sound waves orthe use of a volatile. While not wishing to be bound by any theory, theuse of elevated pressure may enhance the effect of irradiation on thebiological contaminant(s) or pathogen(s) and/or enhance the protectionafforded by one or more stabilizers, and therefore allow the use of alower total dose of radiation. Suitable pressures can be determinedempirically by one skilled in the art.

[0114] Generally, according to these methods, the pH of the preparationcontaining a biological material undergoing sterilization is about 7. Insome embodiments, however, the preparation containing a biologicalmaterial may have a pH of less than 7, preferably less than or equal to6, more preferably less than or equal to 5, even more preferably lessthan or equal to 4, and most preferably less than or equal to 3. Inalternative embodiments, the preparation containing a biologicalmaterial may have a pH of greater than 7, preferably greater than orequal to 8, more preferably greater than or equal to 9, even morepreferably greater than or equal to 10, and most preferably greater thanor equal to 11. According to certain embodiments, the pH of thepreparation containing a biological material undergoing sterilization isat or near the isoelectric point of one of the components of thepreparation containing a biological material. Suitable pH levels can bedetermined empirically by one skilled in the art.

[0115] Similarly, according to these methods, the irradiation of thepreparation containing a biological material may occur under anyatmosphere that is not deleterious to the preparation containing abiological material being treated. According to one preferredembodiment, the preparation containing a biological material is held ina low oxygen atmosphere or an inert atmosphere. When an inert atmosphereis employed, the atmosphere is preferably composed of a noble gas, suchas helium or argon, more preferably a higher molecular weight noble gas,and most preferably argon. According to another preferred embodiment,the preparation containing a biological material is held under vacuumwhile being irradiated. According to a particularly preferredembodiment, the preparation containing a biological material(lyophilized, liquid or frozen) is stored under vacuum or an inertatmosphere (preferably a noble gas, such as helium or argon, morepreferably a higher molecular weight noble gas, and most preferablyargon) prior to irradiation. According to an alternative preferredembodiment, the preparation containing a biological material is heldunder low pressure, to decrease the amount of gas, particularly oxygenand nitrogen, dissolved in the liquid, prior to irradiation, either withor without a prior step of solvent reduction, such as lyophilization.Such degassing may be performed using any of the methods known to oneskilled in the art. For example, the preparation containing a biologicalmaterial may be treated prior to irradiation with at least one cycle,and preferably three cycles, of being subjected to a vacuum and thenbeing placed under an atmosphere comprising at least one noble gas, suchas argon, or nitrogen.

[0116] The sensitivity of a particular biological contaminant orpathogen to radiation is commonly calculated by determining the dosenecessary to inactivate or kill all but 37% of the agent in a sample,which is known as the D₃₇ value. The desirable components of apreparation containing a biological material may also be considered tohave a D₃₇ value equal to the dose of radiation required to eliminateall but 37% of their desirable biological and physiologicalcharacteristics.

[0117] In accordance with certain preferred methods, the sterilizationof the preparation containing a biological material is conducted underconditions that result in a decrease in the D₃₇ value of the biologicalcontaminant or pathogen without a concomitant decrease in the D₃₇ valueof the preparation containing a biological material. In accordance withother preferred methods, the sterilization of the preparation containinga biological material is conducted under conditions that result in anincrease in the D₃₇ value of the preparation containing a biologicalmaterial. In accordance with the most preferred methods, thesterilization of the preparation containing a biological material isconducted under conditions that result in a decrease in the D₃₇ value ofthe biological contaminant or pathogen and a concomitant increase in theD₃₇ value of the preparation containing a biological material.

[0118] In accordance with certain preferred methods, the sterilizationof the preparation containing a biological material is conducted underconditions that reduce the possibility of the production ofneo-antigens. In accordance with other preferred embodiments, thesterilization of the preparation containing a biological material isconducted under conditions that result in the production ofsubstantially no neo-antigens.

[0119] In accordance with certain preferred methods, the sterilizationof the preparation containing a biological material is conducted underconditions that reduce the total antigenicity of the preparationcontaining a biological material. In accordance with other preferredembodiments, the sterilization of preparation containing a biologicalmaterial is conducted under conditions that reduce the number ofreactive allo-antigens and/or xeno-antigens in the preparationcontaining a biological material.

[0120] According to certain preferred embodiments, preparationcontaining a biological material sterilized according to the methodsdescribed herein may be introduced into a mammal in need thereof forprophylaxis or treatment of a condition or disease or malfunction of atissue. Methods of introducing such preparation containing a biologicalmaterial into a mammal are known to those skilled in the art.

[0121] When employed in such embodiments, preparation containing abiological material sterilized according to the methods described hereindo not produce sufficient negative characteristics in the preparationcontaining a biological material following introduction into the mammalto render the preparation containing a biological material unsafe and/orineffective for the intended use thereof. Illustrative examples of suchnegative characteristics include, but are not limited to, inflammationand calcification. Such negative characteristics may be detected by anymeans known to those skilled in the art, such as MRIs, CAT scans and thelike.

[0122] According to particularly preferred embodiments, sterilization ofthe preparation containing a biological material is conducted after thepreparation containing a biological material is packaged, i.e. as aterminal sterilization process.

EXAMPLES Example 1 Paste Experiment

[0123] Purpose: To evaluate dose distribution and temperature profilefor a volume of bovine paste gamma irradiated in an insulated container.In this experiment, an insulated container (24″ H×23.75″W×23.57″L)containing frozen bovine paste (14″ H×10.5″W×10.25″L) and dry ice wasirradiated with gamma irradiation in a carrier batch facility (FIG. 1).Irradiation was carried out in two unequal dose fractions to a specifiedsurface dose. Thermocouples (TC's) and dosimeters were placed throughoutthe paste (FIG. 2). Thermal labels and dosimeters were placed externallyon the insulated container (FIG. 3).

[0124] Equipment and Materials:

[0125] 1. Omega Type T TC's (Stock #5SC-TT-T-36-72); 36 gauge wire, 72″length;

[0126] 2. Omega TC Readers: Model HH202A (Serial No. 20910, reader 1;and Serial No. 20909, reader 2).

[0127] 3. Envirocooler Insulated Container EVC-30-40-LL

[0128] Outer Dimensions: 24″H×23.75″W×23.57″L

[0129] Inner Dimensions: 17.5″H×17.75″W×17.75 ″L

[0130] 4. Cardboard Paste Container: 14″H×10.5″W×10.25″L

[0131] 5. Dry Ice (pellets)

[0132] 6. Bovine Fraction “A” Paste: Filter press paste (50-60% IgG),diatomaceous earth and Perlite, prepared using the Kistler & Nitschmannfractionation procedure

[0133] 7. Dosimeters: Alanine pellets (Gamma Service, Batch T79801) andRed 4034 Perspex GH

[0134] 8. 0.6 ml Microfuge Tubes

[0135] 9. Cardboard Slip Sheets

[0136] 10. Thermal Labels (Paper Thermometer Co., Set #10, 32.3-54.4°C.)

[0137] 11. STERIS Batch-type Irradiator (IR-131), Whippany, N.J.

[0138] Procedure:

[0139] 1. The functionality of the TC's using two readout instrumentswas assessed and each TC was uniquely identified. A 3-point verificationat nominal temperatures of 22° C., 0° C. and −78° C. was used. Only TC'sthat performed to within 2° C. of reference temperature were used.

[0140] 2. Prepared dosimeters. Each dosimeter consisted of three alaninedosimeters in a 0.6 ml Microfuge Tube. Each tube was uniquely labelled.

[0141] 3. Dosimeters and TC's 5 were arranged on three cardboard slipsheets as shown in FIG. 2.

[0142] 4. Envirocooler container was labelled Front, Rear and Top, asshown in FIG. 3.

[0143] 5. Polyethylene bags were cut and glued to the interior surfaceof the paste carton to protect the cardboard surface from excessmoisture.

[0144] 6. As shown in FIG. 2, a first cardboard slip sheet 10 was placedin the bottom of the paste container and the paste container was filledto approximately mid-height with bovine paste. A second cardboard slipsheet 20 was placed on top of the bovine paste and the paste containerwas filled to the top with bovine paste. A third cardboard slip sheet 30was placed on top of the bovine paste. The height of the second slipsheet relative to the first slip sheet was recorded. The height of thethird slip sheet relative to the first and second slip sheets wasrecorded.

[0145] 7. The carton was sealed with the TC leads for each slip sheetbundled together and extending from the container for subsequenttemperature measurements.

[0146] 8. The paste was frozen at −40° C. Once the paste was frozen, thepaste container was measured and weighed.

[0147] 9. Placed the paste container 40 in the Envirocooler 50, as shownin FIG. 1. The bundled TC leads were extended and secured to the surfaceof the Envirocooler.

[0148] 10. Added cardboard sleeves 60 that extend from front to back ofthe Envirocooler 50.

[0149] 11. Placed Styrofoam into the void spaces 80 to stabilizer thepaste container in the Envirocooler 50.

[0150] 12. Weighed the container.

[0151] 13. Placed dry ice between the cardboard sleeves 60 and the sidesof the Envirocooler 50.

[0152] 14. Sealed the container.

[0153] 15. Positioned thermal tape and external dosimeters on the rearface, front face, top and bottom of the Envirocooler, as shown in FIGS.3a-3 c.

[0154] 16. Just prior to irradiation, measured temperature at each TC.

[0155] 17. Placed the container in the carrier on top of about 32″ ofcardboard, such that the front and rear faces of the container wereparallel to the source plaque.

[0156] 18. Placed about 4″ of cardboard on top of the container.

[0157] 19. Irradiated the container to achieve a surface dose of 50 kGy(dose in water) in two unequal dose portions.

[0158] 20. Following each dose portion, recorded the temperature foreach TC.

[0159] 21. After the final dose portion, removed the container from thecarrier, removed external dosimeters, weighed the container and recordedthe thermal label results.

[0160] Results: Weight Summary: Item Weight (lbs.) Envirocooler (empty)17.2 Paste Carton (filled) 42.2 Dry Ice Start 63.6 End 40.0 Total Weight(packed container) 123

[0161] Maximum recorded temperatures ranged from 38° C. to 54° C.(approximately a 15 to 30°0 C. rise for all labels). F ront face labelsranged from 49 to 54° C. while rear face labels ranged from 38 to 43° C.Top and bottom labels read 38° C. Temperature were observed to havereached the maximum reported values during the first radiation dosefraction.

[0162] According to the TC measurements, temperatures within the paste,generally, rose about 15° C. or less. The minimum dose received by thepaste, 37.4 kGy, occurred at the geometric center, while the maximumdose received by the paste, 55.4 kGy, occurred at the rear top centerposition. The dose drop off from the front or rear center position tothe midsection center position was approximately 30% (±15%). Alaninedoses were consistently higher than the corresponding Red 4034 dosevalues.

[0163] The Envirocooler container (3″ thick) and dry ice arrangementprovided temperature control throughout all stages of preparation,irradiation and post-irradiation handling.

Example 2

[0164] Purpose: To test the ability of two devices to maintain thetemperature of an alanine pellet at dry ice temperature while beingexposed to gamma irradiation.

[0165] Materials and Equipment:

[0166] 1. Wheaton l120cc bottles (Catalog # 225546, Lot #1208867-01)with dimension 4″ high ×1.75″ diameter.

[0167] 2. Dry ice blocks and pellets-Artic Ice

[0168] 3. Craftsman cordless power drill

[0169] 4. Cheesecloth

[0170] 5. Hammer

[0171] 6. Glue stick

[0172] 7. Omega Engineering Type T, 36 AWG, 72″ long wire, Tefloninsulated Thermocouples (Catalog #5SC-TT-T-36-72)- numbered I through 4.

[0173] 8. Omega Engineering thermocouple extension cables SMP female toSMP male, 10 ft long (Catalog # TECT-10-9)

[0174] 9. Temperature Recording Device High Precision Temperature andVoltage Meter: National Instruments Model NI 4350, Part # 184374C-01, SNCD2141

[0175] a. Analog Input device: National Instruments Model TC 2190, Part#184473B-10, SN CB2154

[0176] b. Dell laptop computer with Virtual Bench Logger softwareprogram

[0177] 10. Gamma Irradiation Facility: Gammacell 207, NationalInstitutes of Standards and Technology, Radiation Physics Building #245,Gaithersburg, Md.

[0178] 11. Vacuum Dewar Flask- Cole Palmer Stainless Steel Dewar FlaskSeries 3763 Cat# D1000W, 1L volume, 16 cm inner height, 10 cm diameter(provided by NIST)

[0179] 12. Cork cover- Cole Palmer D1000W Cat#03763-32 (provided byNIST)

[0180] Procedure:

[0181] 1. Trimmed excess foil packaging on two (2) alanine pelletblister pack to ˜½″ square.

[0182] 2. Cut block dry ice slab to make a piece with dimensions4″×1.5″×1.5″ (H×D ×W).

[0183] 3. Using a drill, fashioned a ½″ square slot (˜⅛″tall) into oneside of a dry ice block at vertical center (˜2″from the top). Stored ininsulated cooler with dry ice to minimize sublimation prior toirradiation.

[0184] 4. With sample chamber in “up” position (i.e. not irradiating)positioned four (4) thermocouples (TC) junctions (#1 thru #4) into thesample chamber of the gammacell 207 with the plugs accessible to theexternal environment.

[0185] 5. Attached TC extensions and set-up temperature recordingdevice.

[0186] 6. Began temperature data acquisition of TC#1 through #4.

[0187] 7. Using cheesecloth and hammer, pulverized pelleted dry ice intoa fine powder.

[0188] 8. Set-up of“Block Dry Ice” alanine temperature control device:

[0189] a. Using a needle, made a small hole in the alanine blister pack.

[0190] b. Fed TC#1 junction and wire into the alanine blister pack suchthat TC#1 junction was in contact with the alanine pellet.

[0191] c. Positioned alanine blister pack-thermocouple #1 assembly intothe air space created in the block form of dry ice (bubble side facingdown).

[0192] d. Placed block dry ice into vacuum dewar with TC wire extendingout of the top of the dewar.

[0193] 9. Set-up of “Powdered dry ice—Bottle” alanine temperaturecontrol device:

[0194] a. Filled one 120 cc glass bottle with ˜½ powdered dry ice andpacked down using glue stick.

[0195] b. Fed TC#2 junction and wire into the alanine blister pack suchthat TC#2 junction was in contact with the alanine pellet.

[0196] c. Positioned alanine blister pack-thermocouple #2 assembly intothe approximate center of the 120cc bottle (bubble side facing down).

[0197] d. Placed powdered dry ice-bottle assembly into vacuum dewar withTC wire extending out of the top of the dewar.

[0198] 10. Positioned TC#3 inside the dewar such that it would recordthe temperature of the air space (i.e. not in contact with the bottlesurface or block dry ice).

[0199] 11. Placed cork lid onto top of dewar.

[0200] 12. Positioned dewar in the gammacell sample chamber.

[0201] 13. Positioned TC#4 in the sample chamber such that it wouldrecord the temperature of the air space in the sample chamber.

[0202] 14. Allowed TCs to equilibrate for ˜10-15 minutes until TC #1 and#2 stabilized to a reading of approximately −78° C.

[0203] 15. Started irradiation and noted temperatures of all TCs.

[0204] 16. Ended irradiation and noted temperature of all TCs.'

[0205] 17. Total Irradiation time was 6.148 hours. Based on estimateddose rate, total delivered dose was 105.84 kGy.

[0206] 18. Disassembled vacuum dewar and alanine temperature controldevices. Noted the amount of dry ice remaining for each device.

[0207] Results:

[0208] The temperature in the near proximity of the alanine pelletincreased 1.25° C. in the block dry ice device for six hours while thepellet was exposed to approximately 105 kGy of gamma irradiation at adose rate of approximately 17.2 kGy/hr.

[0209] The temperature in the near proximity of the alanine pelletincreased 0.65° C. in the powdered dry ice-bottle device for six hourswhile the pellet was exposed to approximately 105 kGy of gammairradiation at a dose rate of approximately 17.2 kGy/hr.

[0210] At least 50% of the dry ice remained in the devices (either solidor powdered) following gamma irradiation.

[0211] Conclusion:

[0212] The temperature of an alanine dosimeter can be maintained towithin +1.25° C. of its starting dry ice temperature (−78° C.) during asix hour exposure to gamma irradiation (−105 kGy total dose) whenpositioned in the either of a 120cc bottle filled with powdered dry iceor a block of solid dry ice (approx. dimensions 4″×1.5″×1.5″). Atemperature summary is presented in Table 1. TABLE 1 Temperature SummaryChange in Temperature TC Initial Final during # Description TemperatureTemperature Irradiation 1 Block Dry Ice −78.009° C. −76.75° C.  +1.25°C. 2 Powdered Dry Ice- −78.088° C. −77.44° C.  +0.648° C. Bottle 3 DewarAir Space −71.684° C. −66.313° C.   +5.371° C. 4 Sample Chamber Air  27.393° C.   55.84° C. +28.447° C. Space

Example 3

[0213] Purpose: To test the ability of a small glass Dewar to maintainthe temperature of an alanine pellet while being exposed to gammairradiation.

[0214] Meterial and Equipment:

[0215] 1. Vacuum Dewar, Silvered, 22 mm OD×12.6 mm ID—H. S. Martin, Inc.

[0216] 2. Styrofoam plug

[0217] 3. Dry ice blocks and pellets-Artic Ice

[0218] 4. Craftsman cordless power drill

[0219] 5. Plug-cutter—1/2″ Irwin #43908

[0220] 6. 50 ml polypropylene tube

[0221] 7. Harwell Batch AC Alanine pellets, heat sealed individuallyinto foil-laminate pouches.

[0222] 8. Omega Engineering Type T, 36 AWG, 72″ long wire, Tefloninsulated Thermocouples (Catalog #5SC-TT-T-36-72).

[0223] 9. Omega Engineering thermocouple extension cables SMP female toSMP male, 10 ft long (Catalog # TECT- 10-9)

[0224] 10. Bruker EPR Spectrometer (eScan) system

[0225] a. Electronics Unit (Model E2043000, Serial No. 0133)

[0226] b. Magnetic Unit (Model E2044000, Serial No. 0133)

[0227] c. Pellet Probe PHOO19

[0228] 11. Denver M-220 analytical (micro) balance (Serial No. P112332)

[0229] 12. Temperature Recording Device

[0230] a. High Precision Temperature and Voltage Meter: NationalInstruments Model NI 4350, Part # 184374C-01, SN CD2141

[0231] b. Analog Input device: National Instruments Model TC 2190, Part# 184473B-10, SN CB2154

[0232] c. Dell laptop computer with Virtual Bench Logger softwareprogram

[0233] 13. Gamma Irradiation Facility: Gamma cell 207, NationalInstitutes of Standards and Technology, Radiation Physics Building #245,Gaithersburg, MD.

[0234] 14. Vacuum Dewar Flask- Cole Palmer Stainless Steel Dewar FlaskSeries 3763 Cat# D1000W, 1 L volume, 16 cm inner height, 10 cm diameter(provided by NIST)

[0235] 15. Cork cover- Cole Palmer D1000W Cat# 03763-32 (provided byNIST)

[0236] Procedure:

[0237] 1. Pre-cooled micro Dewar in dry ice for 30-60min.

[0238] 2. Taped alanine pellet in foil-laminate pouch onto outside ofDewar.

[0239] 3. With sample chamber in “up” position (i.e. not irradiating)positioned three (3) thermocouples (TC) junctions (#1, #2, and #3) intothe sample chamber of the gamma cell 207 with the plugs accessible tothe external environment.

[0240] 4. Attached TC extensions and set-up temperature recordingdevice.

[0241] 5. Began temperature data acquisition.

[0242] 6. Pre-cooled the stainless stain Dewar by filling it withpelleted dry ice (˜30min)

[0243] 7. Prepared solid dry ice “plugs” for use in the Dewar.

[0244] 8. Using lab tape, secured TC#1 with an alanine pellet infoil-laminate pouch bottle surface or block dry ice.

[0245] 9. Filled the micro Dewar ˜1/2 full with dry ice plugs.

[0246] 10. Place alanine pellet/TC#1 packet into the Dewar and tappeddown using a pen to make contact with the dry ice.

[0247] 11. Added dry ice pellets on top of alanine pellet/TC#1 andtapped down using a pen.

[0248] 12. Covered the Dewar opening with a Styrofoam plug.

[0249] 13. Placed Dewar in 50 ml polypropylene tube.

[0250] 14. Secured TC#2 in the air space between the micro Dewar and the50 ml tube.

[0251] 15. Placed cork lid onto top of stainless steel Dewar.

[0252] 16. Positioned stainless steel Dewar in the gamma cell samplechamber.

[0253] 17. Allowed TCs to equilibrate for ˜10-15 minutes until TC #1 and#2 stabilized to a reading of approximately −78° C.

[0254] 18. Started irradiation and noted temperature of all TCs.

[0255] 19. Ended irradiation and noted temperature of all TCs.

[0256] 20. Total irradiation time was 5 hours, 53 minutes, 34 seconds.Based on estimated dose rate, total delivered dose was 99.23 kGy.

[0257] 21. Disassembled vacuum Dewar and alanine temperature controldevices. Noted the amount of dry ice remaining in the micro Dewar.

[0258] 22. Compute the absorbed dose to both alanine pellets.

[0259] Results:

[0260] The temperature in the near proximity of the alanine pelletincreased 1.9° C. in the ar while the pellet was exposed toapproximately six hours of gamma irradiation at a of approximately 16.8kGy/hr. Approximately 50% of the dry ice remained in the ar devicefollowing gamma irradiation.

[0261] Conclusions:

[0262] The temperature of an alanine dosimeter can be maintained towithin 2° C. of its starting dry ice temperature (−78° C.) during sixhours exposure to gamma irradiation (˜100 kGy total dose) whenpositioned in the center of a small glass vacuum Dewar. A temperature ispresented in Table 2. TABLE 2 Temperature Summary Change in AverageTemperature Temperature TC Descrip- Initial Final during during the #tion Temperature Temperature Irradiation irradiation 2 Alanine  −78.12°C. −76.227° C. +1.9° C. −77 Pellet inside Dewar 3 Alanine −78.992° C.−73.877° C. +5.1° C. −76.005 Pellet outside Dewar

Example 4

[0263] Purpose: To perform qualitative and quantitative analyses ofHarwell alanine pellet dosimeters with respect to reproducibility(evaluating replicate samples irradiated to known dose levels),calibration (determination of dose response at fixed temperature), anddose response when irradiated at low temperatures.

[0264] Materials and Equipment:

[0265] A. Harwell alanine pellet dosimeters

[0266] B. Microfuge tubes

[0267] C. Polypropylene “snap-cap” tubes

[0268] D. Bruker EPR spectrometer (eScan) system

[0269] 1. Electronics Unit (Model E2043000, Serial No.0133)

[0270] 2. Magnetic Unit (Model E2044000, Serial No.0133)

[0271] 3. PelletprobePH00019

[0272] E. Denver M-220 analytical (micro)balance (Ser. No. P112332)

[0273] F. NIST Gammacell 220 irradiator(GC207) and alanine pellet holder

[0274] G. NIST temperature controller and recorder

[0275] H. Dickson Temperature and Relative Humidity data logger (TP120,Ser. No. 02222169)

[0276] Experiment Details:

[0277] A. Sample Handling, Labeling and Storage

[0278] 1. Individual dosimeters were stored in a polymer canister.

[0279] b. Samples were provided to NIST for irradiation and stored (pre-and post-irradiation) in accordance with NIST's standard practices.Following irradiation:

[0280] a. Individual pellets were identified with a sample number;

[0281] b. Calibration samples were individually stored in microfugetubes.

[0282] c. Temperature samples were stored in polypropylene (snap-cap)tubes. All samples for a given dose and irradiation temperature werestored in a single tube.

[0283] 2. Upon return from NIST, calibration and temperature dosimeterswere identified according to

[0284] d. Dosimeter manufacturer,

[0285] e. Dosimeter batch,

[0286] f. Nominal dose,

[0287] g. Nominal irradiation temperature, and

[0288] h. Dosimeter number (for replicate samples).

[0289] B. Irradiation Studies—Calibration and Temperature

[0290] 1. Dosimeters were irradiated according to the irradiationschedule in Table 1

[0291] 2. Calibration samples consisted of four (4) dosimeters at eachdose point plus controls.

[0292] b. Temperature samples consisted of six (6) dosimeters at eachdose point plus controls.

[0293] 1. All dosimeters to be used for the calibration and temperaturestudies were visually inspected. No samples needed to be replaced as aresult of this inspection

[0294] C. EPR Analysis of Dosimeters

[0295] 1. Dosimeters were allowed to equilibrate to room conditions fora minimum of 24 hours following the end of irradiation before readingthem.

[0296] 2. The mass of each pellet was taken after EPR analysis wasperformed

[0297] 3. To assess any change in dosimeter response with time,calibration and selected temperature samples were reanalyzed duringsubsequent sample analysis sessions for a period of at least one monthfollowing irradiation. TABLE 1 Irradiation Schedule Nominal dose NominalIrradiation temperature (° C.) (kGy, water) −75 −55 −45 −35 −25 −10 0 515 25* 25 35 50 0.5 X X X X X X X X X X X X 5 X X X X X X X X X X X X X10 X 25 X X X X X X X X X X X X 40 X 50 X X X X X X X X X X X X X 55 X60 X X X X X X X X 70 X 80 X X X X X X X X X X X X 85 X 100 X X X X X XX X 115 X 130 X

Results and Data Analysis

[0298] Calibration data for EPR measurements showed high precision amongreplicate samples at each dose point. The highest % CV value, 0.55%, wasat 85 kGy. This same trend occurred when these same dosimeters were readagain about one or two months later. FIG. 4 illustrates thecomparability of average (mean) measurements over a 3-month period fordosimeters irradiated at various temperatures to various total doses.Sample masses were consistent with the manufacturer's batch massdistribution range (59.5 to 61.2 mg).

[0299] Irradiation temperature varied for each group of dosimetersamples, ranging from 22.6 to 26.2° C., with an average of 24.4±1.1° C.By convention, 25° C. was used as the calibration reference temperature.

[0300]FIG. 5 is a plot of the mass-corrected dosimeter response(Ratio/mg) vs. absorbed dose over the dynamic calibration range foralanine dosimeters irradiated with gamma irradiation. FIG. 5 illustratesa non-linear relationship.

[0301] Inverting the axes, we developed a mathematical function thatdescribes the dose-response, D(R), relationship. A calibration curve(for fixed temperature T=25° C.) was developed covering the range 25 to115 kGy. (See FIG. 6). The resulting equation is expressed as follows:

D(R)=22676188.37R ³−552494.84R ²+9102.47R−22.24

[0302] where

[0303] D=dose in kilograys (water), and

[0304] R=mass-corrected response at 25° C.

[0305] Coefficients have been rounded to 2 decimal places.

[0306] Besides observing a high R² value (indicating a high correlationbetween the variables), the suitability of the expression was assessedby substituting the mass-corrected dosimeter response into theanalytical equation in FIG. 6. These dose ratios ranged from 0.984 to1.018 (or −1.6% to +1.8%).

[0307] Temperature data and analysis for doses of 5, 25, 50, 60, 80 and100 kGy. demonstrate high precision among replicate samples at each dosepoint. The highest % CV value, 1.88%, occurred at 100 kGy.

[0308]FIG. 7 is a plot of relative dosimeter response (relative to themean dosimeter response at 25° C.) vs. irradiation temperature for eachdose.

[0309] At dry ice temperatures, the dosimeter response (relative to 25°C.) decreases by approximately 23% to 25% for doses of 5 and 25 kGy, andby approximately 30% to 35% for doses of 50, 60, 80 and 100 kGy. Atfixed dose, the dosimeter response as a function of irradiationtemperature is predictable and can be described by a mathematicalrelationship. This mathematical relationship is dose-dependent,especially below approximately −10° C.

[0310] The suitability of each of the mathematical expressions shown inFIG. 7 was assessed by substituting the irradiation temperature into theanalytical equation corresponding to the specified dose to obtain apredicted relative response. The predicted relative response wascompared to the actual relative response by dividing one quantity by theother. Limiting the assessment to two representative expressions, theratio at 50 kGy ranged from 0.986 to 1.013 (or −1.4% to +1.3%), and theratio at 60 kGy ranged from 0.987 to 1.011 (or −1.3% to +1.1%).

[0311] To determine doses in the range 25-115 kGy and for irradiationtemperatures (T) covering the range of −78 to +50° C., the measuredresponse (R_(M))_(T° C.) was first divided by a temperature correctionfactor relative to 25° C. (CF)_(25° C.):

R _(25° C.)=(R _(M))T° C./(CF)_(25° C.):

[0312] and then the dose response equation, D(R), was applied.

[0313] To test this approach, we determined the temperature-correctedresponse, R_(25° C.), for the 50 kGy temperature data, and then computedthe dose using the dose response equations, supra.

[0314] The predicted dose was compared to the experimentally determineddose by dividing one quantity by the other to get a ratio of doses. Thedose ratio at 50 kGy over the temperature range ranged from 0.976 to1.014 (or −2.6% to +1.4%).

[0315] As noted above, the CF value is dose-dependent. Practically, itis preferable to develop a single temperature correction (function) thatcovers as broad a dose range as possible. To assess the ability toaccurately predict the dose using dose range-based CF values,calibration data were combined into dose ranges—50 & 60 kGy; 50, 60 & 80kGy; 60 & 80 kGy; and 80 & 100 kGy—and plotted (see FIG. 8).

[0316] The resulting equations were used to compute the R_(25° C.) andthe absorbed dose using the calibration curve above. FIG. 9 illustratesthe comparison of computed dose to the reported dose (for each of thefour dose ranges) as a function of irradiation temperature.

[0317] a. 50 and 60 kGy and T=−78 to +50° C.:

[0318] CF=0.000000133T³−0.000016354T²+0.001539890T+0.972303706 Doseratios Minimum: 0.96 Maximum: 1.03 Mean ± SD: 0.993 ± 0.013

[0319] b. 60 and 80 kGy and T=−78 to +35° C.:

[0320] CF=0.0000000427T³−0.0000243867T²+0.0017031262T+0.9757045570 Doseratios Minimum: 0.97 Maximum: 1.03 Mean +/− SD: 1.001 ± 0.013

[0321] c. 50, 60 and 80 kGy and T=−78 to +50° C.:

[0322] CF=0.000000104T³−0.000018740T²+0.001637463T+0.973579951 Doseratios Minimum: 0.95 Maximum: 1.05 Mean +/− SD: 0.997 ± 0.017

[0323] d. 80 and 100 kGy and T=−78 to +35° C.:

[0324] CF=0.0000000611T³−0.0000265639T²+0.0017373535T+0.9786412509 Doseratios Minimum: 0.94 Maximum: 1.06 Mean +/− SD: 1.007 ± 0.022

[0325] Considering the broadest dose range, 50 to 80 kGy, computed andexperimentally obtained doses agreed generally to within about 3%.

[0326] As noted above, the temperature response appears to be doseindependent above approximately −10° C. This is illustrated in FIG. 10where the response as a function of temperature increases linearly,having a slope ˜+0.0011, or +0.11%/° C. over the dose range of 5 to 100kGy. Thus, over the range of −10 to +50° C., a scalar temperaturecorrection factor can be applied that does not depend on the dose.

[0327]FIG. 11 shows a scatter plot (response vs. dose) at dry icetemperature (−77.4 to −75.4° C.) over the dose range of 25 to 100 kGy.The logarithmic equation, Response=0.00461601n (Dose)−0.0092011, caneasily be translated to develop a dose(D)-response(R) relationship asfollows:

D(R)=e ^(((R+0.0092011)/0.0046160))

[0328] Using this equation to compute the dose, and conducting a similaranalysis of the computed (fitted) and reported (actual) doses, thefollowing was observed regarding the dose ratios: Dose ratios Minimum:0.95 Maximum: 1.05 Mean +/− SD: 1.000 ± 0.023

[0329] The average dose ratios for each dose level ranged from 0.98 to1.02 (i.e. agreement within 2%) with the exception of 80 kGy data, whereratio was 1.037. Thus, doses computed using this low-temperaturecalibration curve provides agreement with reported doses to within 4%.

Conclusions

[0330] These dosimeters may be used for measuring absorbed doses asshown below:

[0331] Based upon calibration data in the range of 25-115 kGy, dose (D)at fixed temperature (T=25° C.) within this range can be accuratelypredicted using the following mathematical expression:

D(kGy, water)_(25° C.) =22676188.3743057R _(25° C.) ³−552494.8437186R_(25° C.) ²+9102.4775300R _(25° C.) −22.2431768

[0332] where R_(25° C.)=mass-corrected response (mg⁻¹) for irradiationtemperature(T)=25° C.

[0333] Alternately, if temperature during irradiation is controlled tomaintain dry ice temperature (−77.4 to −75.4° C.), the followingmathematical expression can be used:

D(kGy, water)_(25° C.) =e ^(((R+00092011)/0.0046160))

[0334] where R=mass-corrected response (mg³¹ ¹) for irradiationtemperature(T)=−78° C.

[0335] At fixed dose, the dosimeter response as a function ofirradiation temperature is predictable and can be described by amathematical relationship. This relationship appears linear fortemperatures above −10° C. and becomes non-linear for temperatures belowapproximately −10° C. Considering the entire temperature range, thismathematical relationship is dose-dependent. However, above ˜−10° C. therelationship appears dose-independent, having a correction factor ofapproximately +1.1%/° C.

[0336] Perform the following generalized steps to determine absorbeddoses based on irradiation temperatures in the range of −78 to +50° C.Correct the measured response (R_(M))_(T° C.) to its corresponding valueat the reference temperature of +25° C. (R_(25° C.)) using theappropriate temperature correction factor (CF):

R _(25° C.)=(R _(M))_(T° C.) /CF

[0337] i. For irradiation temperature (T) below −10° C. and dose range50-80 kGy,

CF=0.000000104T ³−0.000018740T ²+0.001637463T+0.973579951

[0338] ii. For irradiation temperature (T) below −10° C. and dose rangeabove 80 kGy and less than 90 kGy,

CF=0.0000000209T ³−0.0000265220T ²+0.0017614971T+0.9775254341

[0339] iii. For irradiation temperature (T) below −10° C. and dose rangeabove 90 kGy and less than 100 kGy,

CF=0.0000000905T ³−0.0000275970T ²+0.0017245754T+0.9808458732

[0340] iv. For irradiation temperature (T) equal to or above −10° C.(dose-independent),

CF=1+[(T−25 )(0.0011)]

[0341] Having now fully described this invention, it will be understoodto those of ordinary skill in the art that the methods of the presentinvention can be carried out with a wide and equivalent range ofconditions, formulations and other parameters without departing from thescope of the invention or any embodiments thereof.

[0342] All patents and publications cited herein are hereby fullyincorporated by reference in their entirety. The citation of anypublication is for its disclosure prior to the filing date and shouldnot be construed as an admission that such publication is prior art orthat the present invention is not entitled to antedate such publicationby virtue of prior invention.

What is claimed is
 1. A device for measuring the amount of energyabsorbed by a product undergoing sterilization with radiation,comprising: (i) an effective amount of at least one material thatabsorbs radiation in a quantifiable manner; and (ii) an effective amountof at least one cooling agent for maintaining said material within apredetermined temperature range between −120° C. and ambient temperatureduring irradiation.
 2. The device according to claim 1, wherein saidmaterial that absorbs radiation is selected from the group consisting ofalanine, cellulose acetate, ethanol chlorobenzene and radiochromicfilms.
 3. The device according to claim 1, wherein said cooling agentcomprises dry ice.
 4. The device according to claim 1, wherein saidcooling agent is of sufficient volume to contain at least a portion ofsaid material that absorbs radiation.
 5. The device according to claim1, wherein said cooling agent is in the form of particles.
 6. The deviceaccording to claim 5, wherein said particles have an average volume ofnot more than 17 cm³.
 7. The device according to claim 5, wherein saidparticles have an average volume of not more than 1 cm³.
 8. The deviceaccording to claim 1, wherein said cooling agent is in the form of asolid or semi-solid.
 9. The device according to claim 1, furthercomprising a container of sufficient volume to contain at least aportion of said cooling agent and at least a portion of said material.10. The device according to claim 9, wherein said container is a vacuumDewar.
 11. A method for determining the amount of energy absorbed by aproduct undergoing irradiation, comprising: (a) placing within asuitable container at least one product to be sterilized and at leastone device comprising: (i) at least one material that absorbs radiationin a quantifiable manner; and (ii) an effective amount of at least onecooling agent for maintaining said material within a predeterminedtemperature range between −120° C. and ambient temperature duringirradiation; (b) irradiating said container containing said product andsaid device; and (c) analyzing said material to determine the amount ofenergy absorbed during irradiation.
 12. A method for maintaining thetemperature of a product undergoing irradiation within a predeterminedtemperature range between −120° C. and ambient temperature, comprising:(a) placing at least one product to be irradiated in a suitablecontainer having at least one side and a bottom, wherein the volumedefined by said container is greater than the volume of said product;(b) placing an effective amount of at least one cooling agent in saidcontainer between said product and said at least one side; and (c)irradiating said container containing said product and said coolingagent with ionizing radiation.
 13. The method according to claim 12,wherein said product comprises a biological material.
 14. The methodaccording to claim 12, wherein said product comprises a material thatabsorbs radiation in a quantifiable manner.
 15. The method according toclaim 11 or 12, wherein said cooling agent comprises dry ice.
 16. Themethod according to claim 11 or 12, wherein said cooling agent is in theform of particles.
 17. The method according to claim 16, wherein saidparticles have an average volume of not more than 17 cm³.
 18. The methodaccording to claim 17, wherein said particles have an average volume ofnot more than 1 cm³.
 19. The method according to claim 11 or 12, whereinsaid cooling agent is in the form of a solid or semi-solid.
 20. Themethod according to claim 11 or 12, wherein said container is a vacuumDewar.
 21. The method according to claim 11 or 12, wherein saidcontainer has a front side and a back side and a first side and a secondside.
 22. The method according to claim 21, wherein said container is afoam box.
 23. The method according to claim 21, wherein said coolingagent is placed between said product and said first side and/or betweensaid product and said second side.
 24. The method according to claim 14,wherein said material is selected from the group consisting of alanine,cellulose acetate, ethanol chlorobenzene and radiochromic films.
 25. Themethod according to claim 11 or 12, wherein said product is frozen. 26.The method according to claim 11 or 12, wherein each endpoint of saidtemperature range is less than ambient temperature.
 27. The methodaccording to claim 11 or 12, wherein at least one endpoint of saidtemperature range is less than the freezing point of said product. 28.The method according to claim 11 or 12, wherein each endpoint of saidtemperature range is less than the freezing point of said product. 29.The method according to claim 11 or 12, wherein at least one endpoint ofsaid temperature range is less than −20° C.
 30. The method according toclaim 11 or 12, wherein each endpoint of said temperature range is lessthan −20° C.
 31. The method according to claim 11 or 12, wherein atleast one endpoint of said temperature range is less than −40° C. 32.The method according to claim 11 or 12, wherein each endpoint of saidtemperature range is less than −40° C.
 33. The method according to claim11 or 12, wherein at least one endpoint of said temperature range isless than −60° C.
 34. The method according to claim 11 or 12, whereineach endpoint of said temperature range is less than −60° C.
 35. Themethod according to claim 11 or 12, wherein at least one endpoint ofsaid temperature range is less than −70° C.
 36. The method according toclaim 11 or 12, wherein each endpoint of said temperature range is lessthan −70° C.
 37. The method according to claim 11 or 12, wherein atleast one endpoint of said temperature range is less than the glasstransition point of said product.
 38. The method according to claim 11or 12, wherein each endpoint of said temperature range is less than theglass transition point of said product.
 39. The method according toclaim 11 or 12, wherein said predetermined range is less than theincrease in temperature that would occur under adiabatic conditions. 40.The method according to claim 11 or 12, wherein said temperature rangeis less than 10° C.
 41. The method according to claim 11 or 12, whereinsaid temperature range is less than 5° C.
 42. The method according toclaim 11 or 12, wherein said temperature range is less than 2° C. 43.The method according to claim 11 or 12, wherein said temperature rangeis less than 1.25° C.
 44. The method according to claim 11 or 12,wherein said temperature range is less than 0.65° C.
 45. The methodaccording to claim 11 or 12, wherein said temperature range is less than0.25° C.
 46. The method according to claim 11 or 12, wherein saidtemperature range is less than 0.1° C.
 47. The method according to claim11 or 12, wherein said temperature range is less than 0.1° C. per kGy ofradiation.
 48. The method according to claim 11 or 12, wherein saidtemperature range is less than 0.05° C. per kGy of radiation.
 49. Themethod according to claim 11 or 12, wherein said temperature range isless than 0.02° C. per kGy of radiation.
 50. The method according toclaim 11 or 12, wherein said temperature range is less than 0.0125° C.per kGy of radiation.
 51. The method according to claim 11 or 12,wherein said temperature range is less than 0.0065° C. per kGy ofradiation.
 52. The method according to claim 13, wherein said biologicalmaterial is selected from the group consisting of dextrose, antithrombinIII, plasma, plasminogen, urokinase, thrombin, trypsin, purified proteinfraction, blood, blood cells, alpha 1 proteinase inhibitor, digestiveenzymes, blood proteins and tissue.
 53. The method according to claim52, wherein said tissue is selected from the group consisting of heartvalves, ligaments and demineralized bone matrix.
 54. The methodaccording to claim 52, wherein said digestive enzymes are selected fromthe group consisting of galactosidases and sulfatases.
 55. The methodaccording to claim 52, wherein said blood proteins are selected from thegroup consisting of albumin, Factor VIII, Factor VII, Factor IV,fibrinogen, monoclonal immunoglobulins and polyclonal immunoglobulins.56. The method according to claim 52, wherein said tissue is selectedfrom the group consisting of tendons, nerves, bone, teeth, bone marrow,skin grafts, cartilage, corneas, arteries, veins and organs fortransplantation.
 57. The method according to claim 13, wherein saidirradiating comprises exposing said biological material to a suitableionizing radiation for a time effective to sterilize said biologicalmaterial.
 58. The device according to claim 11 or 12, wherein eachendpoint of said temperature range is less than ambient temperature. 59.The device according to claim 1, wherein at least one endpoint of saidtemperature range is less than the freezing point of said product. 60.The device according to claim 1, wherein each endpoint of saidtemperature range is less than the freezing point of said product. 61.The device according to claim 1, wherein at least one endpoint of saidtemperature range is less than −20° C.
 62. The device according to claim1, wherein each endpoint of said temperature range is less than −20° C.63. The device according to claim 1, wherein at least one endpoint ofsaid temperature range is less than −40° C.
 64. The device according toclaim 1, wherein each endpoint of said temperature range is less than−40° C.
 65. The device according to claim 1, wherein at least oneendpoint of said temperature range is less than −60° C.
 66. The deviceaccording to claim 1, wherein each endpoint of said temperature range isless than −60° C.
 67. The device according to claim 1, wherein at leastone endpoint of said temperature range is less than 70° C.
 68. Thedevice according to claim 1, wherein each endpoint of said temperaturerange is less than −70° C.
 69. The device according to claim 1, whereinat least one endpoint of said temperature range is less than the glasstransition point of said product.
 70. The device according to claim 1,wherein each endpoint of said temperature range is less than the glasstransition point of said product.
 71. The device according to claim 1,wherein said predetermined range is less than the increase intemperature that would occur under adiabatic conditions.
 72. The deviceaccording to claim 1, wherein said temperature range is less than 10° C.73. The device according to claim 1, wherein said temperature range isless than 5° C.
 74. The device according to claim 1, wherein saidtemperature range is less than 2° C.
 75. The device according to claim1, wherein said temperature range is less than 1.25° C.
 76. The deviceaccording to claim 1, wherein said temperature range is less than 0.65°C.
 77. The device according to claim 1, wherein said temperature rangeis less than 0.25° C.
 78. The device according to claim 1, wherein saidtemperature range is less than 0.° C.
 79. The device according to claim1, wherein said temperature range is less than 0.1° C. per kGy ofradiation.
 80. The device according to claim 1, wherein said temperaturerange is less than 0.05° C. per kGy of radiation.
 81. The deviceaccording to claim 1, wherein said temperature range is less than 0.02°C. per kGy of radiation.
 82. The device according to claim 1, whereinsaid temperature range is less than 0.0125° C. per kGy of radiation. 83.The device according to claim 1, wherein said temperature range is lessthan 0.0065° C. per kGy of radiation.